Methods and devices for nucleic acid purification

ABSTRACT

The invention provides pipette tip columns and automated methods for the purification of nucleic acids including plasmids. Nucleic acids can be purified from unclarified, clarified or partially-clarified cell lysates that contain cell debris. The columns typically include a bed of medium positioned in the pipette tip column, above a bottom frit and with an optional top frit. Plasmid preparation scales include miniprep, midiprep, maxiprep, megaprep and gigaprep.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 14/585,070, filed Dec. 29, 2014, which is a continuation inpart of U.S. patent application Ser. No. 13/434,656 filed Mar. 29, 2012,which is a continuation in part of International Application No.PCT/US11/30232 filed Mar.29, 2011, the disclosures of which areincorporated herein by reference in their entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to methods and devices for sample preparation,such as separating (i.e., extracting or purifying) nucleic acids such asDNA and RNA, including circular self-replicating elements such asplasmids, BACs, YACs, cosmids, fosmids and bacteriophage vectors such asM13. Pipette tip columns are used to purify nucleic acids from tissues,unclarified cell lysates or other samples containing particulates andcell debris. Nucleic acids purified by methods of the invention areessentially endotoxin-free, making suitable for mammalian transfectionand transformation. Depending on the scale of the preparation, up to 15mg of plasmid DNA can be obtained in an automated fashion

BACKGROUND OF THE INVENTION

Commercially-available formats for nucleic acid purification includespin columns, magnetic beads in a tube or the use of vacuum to drawliquids through a column or plate. In these formats, nucleic acids areisolated as follows. The cells are grown in a suitable medium, theculture is centrifuged to collect the cells and the growth medium isdiscarded. Next, the cells are lysed to release the nucleic acids.Usually, a second centrifugation step is performed after lysis to pelletthe cell debris and the nucleic acids are purified from the supernatant.

When the spin column format is employed, several additionalcentrifugations are performed. Because these methods require at leasttwo centrifugation steps, they are time-consuming, laborious anddifficult to fully automate. They require significant human interventionand cannot be performed in a walk-away fashion. Therefore, there existsa need for a more automated methods of plasmid and nucleic acidpreparation.

Furthermore, plasmids purified by existing commercially-availablemethods often contain significant amounts of endotoxin, making themunsuitable for transfection. Therefore, there exists a need forautomated, high-throughput nucleic acid purification. Pipette tipcolumns can be used to meet this need. Additionally, there exists a needfor purifying nucleic acids from unclarified cell lysates and othersamples containing particulates and cell debris. For plasmidpurification, there is a need for endotoxin-free plasmid.

Additionally, there exists a need for large-scale automated nucleic acidpreparation. ThermoFisher Scientific offers the BenchPro instrumentdesigned to automatically purify two maxi-scale plasmid preparations.However, the BenchPro instrument can only accommodate up to 125 mL ofbacterial culture grown in LB which is a maximum ofapproximately=1.25×10¹¹ total cells. The maximum yield of plasmid DNAthat can be obtained from the BenchPro 2100 is 1.5 mg. The BenchProcannot accommodate larger volumes or bacterial cultures grown in a richmedium such as Terrific Broth (TB). Therefore, there exists a need forhigher yield, larger-scale, automated plasmid preparations. Furthermore,since the BenchPro 2100 is limited to the maxiprep scale, there exists aneed for instrument that can perform megaprep and gigaprep plasmidpurifications

SUMMARY OF THE INVENTION

An automatable method for purifying nucleic acids in a pipette tipcolumn format was developed. Nucleic acids purified by the methods ofthe invention are substantially endotoxin-free and thereby, suitable fortransfection. In some embodiments, nucleic acids are purified after thecell lysis step without the need for cell debris removal. In theseembodiments, nucleic acids can be purified directly from an unclarifiedlysate in an automated fashion.

In other embodiments, cell debris can be partially removed using apartial filtration process. In still other embodiments, cell debris canbe completely removed. The method is well suited for purification ofplasmids and genomic DNA.

In some embodiments, the plasmid purification process can be scaled upto maxi, mega and gigaprep. In these embodiments, up to 2 L bacterialcultures can be processed using a 20 mL pipette tip column that containsup to 10 mL of resin to obtain up to 15 mg of plasmid DNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an embodiment of the pipette tip column.

FIGS. 2A-2C depict an embodiment of the drying station with front andside views.

FIGS. 3A-3B depict an embodiment of the drying station block with topand bottom views.

FIG. 4 depicts the layout of the deck of the Tecan Freedom Evo automatedliquid handler.

FIGS. 5A-5B is a flowchart comparison of invention versus existingmethods.

FIG. 6 depicts a cut-away view of one embodiment of the partialfiltration apparatus.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to methods and devices for extracting nucleicacids, including plasmids from a sample solution. In U.S. patentapplication Ser. No. 10/620,155, now U.S. Pat. No. 7,482,169,incorporated by reference herein in its entirety, methods and devicesfor performing low dead column extractions are described. In U.S. patentapplication Ser. No. 12/767,659, also incorporated by reference hereinin its entirety, columns and methods for purification of DNA vectors aredescribed.

It is a goal of the instant invention to develop an automated method fornucleic acid purification and plasmid preparation. In certainembodiments, these methods are performed in a pipette tip column format.In some embodiments, a high-throughput method is desirable. Commonlyused commercially-available formats for nucleic acid purificationinclude spin columns, vacuum plates and test tubes.

In the invention described herein, nucleic acids can be purified fromany source. In some embodiments, they can be purified from biologicalsources such as cells. The cells from which nucleic acids are isolatedcan be eukaryotic or prokaryotic. In certain embodiments, plasmids canbe purified from a mixture of nucleic acids or from a gel. Excellentyield and concentration can be obtained using this method. For example,a yield of up to 30 μg of plasmid DNA can be obtained from a 1.4-mL E.coli culture. Plasmid DNA isolated using the methods described herein issubstantially endotoxin-free.

Nucleic acids can be purified in an automated fashion. In certainembodiments, plasmids can be purified from multiple samplessimultaneously with a robotic workstation or electronic pipette.Typically, automated methods are performed with pipette tips and 96-wellplates arranged in a 9 mm center-to-center format. However, otherformats are possible, e.g., 4.5 mm center-to-center or 18 mmcenter-to-center. In some embodiments, multiple purifications areperformed simultaneously in an automated fashion whereas in otherembodiments, a one or two samples can be purified. Plasmid DNA can bepurified in an automated fashion from up to 2 L of bacterial culture.

It is a goal of the invention is to reduce the number of manualprocessing steps used for purifying nucleic acids. That is, it isdesirable to perform separations with minimal operator intervention.

In certain embodiments, the automated purification procedure begins withresuspension of the cell pellet. That is, nucleic acids are purifieddirectly from an unclarified lysate in an automated fashion (FIG. 5B).There is no need for cell debris removal. Because the method isperformed on an unclarified lysate, fewer disposables are needed andconsequently, the cost is lower.

Although it was desirable to eliminate the cell debris removal step andisolate nucleic acids directly from an unclarified lysate, it wastechnically quite difficult to accomplish. Pipette tip columns provide aunique set of technical challenges not present in other formats such asspin columns or vacuum plates. One challenge is the pump. When using aliquid handling robot, the pressure available to push liquids throughthe columns is very low compared to centrifugation or vacuum. A secondchallenge is the volume constraints imposed by the pipette tip columnformat and the use of 96-well plates.

In addition, the unclarified lysate is much more heterogeneous, viscousand gelatinous than a clarified lysate. It contains all cellularcontents including cell debris, genomic DNA, particulates and liquid. Itis surprising that plasmid DNA can be effectively purified from such aheterogeneous mixture.

In other embodiments, the unclarified lysate is subjected to partialfiltration prior to nucleic acid isolation. Partial filtration yields apartially-clarified lysate. In still other embodiments, nucleic acidscan be purified from a clarified lysate. The clarified lysate can beobtained by centrifugation or filtration.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific embodimentsdescribed herein. It is also to be understood that the terminology usedherein for the purpose of describing particular embodiments is notintended to be limiting. As used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to polymer bearing a protected carbonyl would include apolymer bearing two or more protected carbonyls, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, specific examples ofappropriate materials and methods are described herein.

Definitions

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

Automated methods are defined herein as methods not requiring humaninteraction.

The term “bed volume” as used herein is defined as the volume of mediumor solid phase within the column.

The term “interstitial volume” of the bed refers to the volume of thebed of extraction medium that is accessible to solvent, e.g., aqueoussample solutions, wash solutions and desorption solvents. This includesthe space between the beads as well as any volume taken up by the poreswithin the beads. The interstitial volume of the bed represents theminimum volume of liquid required to saturate the column bed.

The term “dead volume” as used herein with respect to a column isdefined as the interstitial volume of the extraction bed, membrane orfrits, and passageways in a column.

Endotoxin is defined as lipopolysaccharide or LPS, a cell wall componentof all gram-negative bacteria such as E. coli. The term, “substantiallyendotoxin-free” is defined herein as an endotoxin concentration of 100EU/μg or less.

The term, “flocculent” is defined herein as a precipitate comprised ofcell debris and reagents formed after the addition of the lysis andprecipitation buffers to the bacterial cells. It has a loosely-clumpedtexture and can have wool-like appearance.

The term “elution volume” as used herein is defined as the volume ofdesorption or elution liquid into which the analytes are desorbed andcollected. The terms “desorption solvent”, “elution liquid”,combinations thereof and the like are used interchangeably herein. Theterm “frit” as used herein is defined as porous material for holding themedium in the column.

The term “pipette tip column” as used herein is defined as any columncontaining a solid phase that can engage a pipette, syringe or liquidhandler, either directly or indirectly. The term, “pipette tip column”is not limited to columns manufactured in pipette tips. Rather, thecolumn can have any shape or geometry as long as it is capable ofengaging a pipette, syringe pump or liquid handling robot. Pipette tipcolumns can be positioned in a rack or incorporated into a plate.

The term “lysis” or “lysed” is a process by which cell are treated tobreak the cell wall and release the nucleic acids.

The term, “plasmid” is defined as an extra-chromosomal, self-replicatingnucleic acid molecule. A plasmid can be a single or double stranded andcan be comprised of DNA or RNA. Cosmids, fosmids, BACs and YACs areconsidered to be within the purview of the plasmid definition.

The term, “unclarified lysate” refers to a cell suspension which hasbeen subjected to lysis.

The term, “gentle mixing” or “gentle pipette mixing” refers toaspiration/expulsion cycles at a flow rate in the range of 0.1 ml/min-10ml/min using a wide-bore pipette.

The term, “cycle” as used herein is defined as a single aspirate/expelstep.

Purification of DNA from E. coli

When purifying plasmid DNA from E. coli, the first step is cell growth.A person of skill in the art can select the appropriate medium dependingon the cell type, number of samples, desired yield, etc. Culture mediacan be chosen based on the bacterial strain. A chemically-defined(synthetic) medium is one in which the exact chemical composition isknown. A rich or complex (undefined) medium is one in which the exactchemical constitution of the medium is not known. Defined media areusually composed of pure biochemicals off the shelf; complex mediausually contain complex materials of biological origin such as blood ormilk or yeast extract or beef extract, the exact chemical composition ofwhich is obviously undetermined. Complex media usually provide the fullrange of growth factors that may be required by an organism so they maybe more handily used to cultivate unknown bacteria or bacteria whosenutritional requirement are complex (i.e., organisms that require a lotof growth factors, known or unknown). Generally, a rich or complexmedium is used such as Terrific Broth, SOC, 2×YT or Agencourt Ale(Beckman Coulter) containing the appropriate antibiotic.

A person of skill in the art can also select the appropriate growthconditions for a given bacterial strain. For a miniprep, bacterial cellscan be grown at 37° C. in a 96-well deep-well block with shaking at 300rpm and harvested in the late logarithmic stage of growth.

Plasmid DNA can be purified from cells grown to a large range of opticaldensities. It is possible to purify plasmid DNA from cells grown to anOD₆₀₀ of 1, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments,plasmid DNA is purified from cells grown to an 0D600 of less than 1greater than 10.

The deep-well block can be selected according to the desired culturevolume. For example, a 4-ml deep well block can be used if a larger cellculture is required. Alternatively, cells can be grown in tubes orflasks if a larger volume is required. After the cells are grown, theyare centrifuged and the growth medium is discarded.

The next step involves resuspension of the cells e.g., in a buffer. Incertain embodiments, the remainder of the procedure can be fullyautomated with the use of a liquid handling system. Generally, mini- andmidi-scale purifications can be automated from the step forward. Maxi-,mega- and gigapreps are often resuspended manually.

In those embodiments in which the procedure is automated, a resuspensionbuffer is added and the cell suspension is repeatedly aspirated andexpelled from a pipette tip until the cells are completely resuspended.Alternatively, the resuspension step may be performed manually byvortexing until the cell pellet is fully resuspended.

After resuspension, the next step is cell lysis. Lysis can beaccomplished by a number of means including physical or chemical action.Non-limiting examples of lysis methods include mechanical, such asultrasonic waves, mortar and pestle, osmotic shock, chemical e.g. bymeans of detergents and/or chaotropic agents and/or organic solvents(e.g. phenol, chloroform, ether), heat and alkali. Lysis via chemicalmeans can be performed on a liquid handling system by addition of alysis solution to the resuspended cells.

When purifying plasmid DNA, a precipitation buffer is added to the lysedcell suspension to precipitate the genomic DNA prior to plasmid capture.In certain embodiments, the precipitation buffer is comprised ofchaotropic salts. Gentle mixing with a wide-bore pipette tip and arelatively low flow rate can be used at this step. After lysis, theplasmid is captured using a pipette tip column.

In existing commercially-available methods, a centrifugation step isusually performed following cell lysis to pellet cell debris. However,an advantage of the instant invention is that this centrifugation stepcan be bypassed making the method considerably more automated than othermethods. In alternate embodiments, the sample can be centrifuged toproduce a clarified lysate are partially filtered to produce a partiallyclarified lysate.

The column can be equilibrated with water or buffer prior to the capturestep. Equilibration can be performed by a single aspiration andexpulsion of water or buffer from the column. After the pipette tipcolumns are equilibrated, DNA can be captured on the equilibrated columnby repeated aspiration and expulsion. In alternate embodiments, thesample is captured using gravity flow. In still other embodiments,nucleic acids are captured using vacuum.

After capture, the columns are usually washed to remove non-specificallybound materials. One or more wash steps can be performed. When more thanone wash is performed, the same wash solution can be used for multiplewashes or different wash solutions can be used. In certain embodiments,the wash solution contains an organic solvent, e.g., alcohol.

Wash steps can be performed with back and forth flow, vacuum orunidirectional flow using gravity or vacuum. The advantage of performingthe wash steps by unidirectional flow is that higher throughput can beachieved. That is, when nucleic acid purification is performed on aliquid handling robot, throughput can be increased by utilizing theliquid handling head simply for dispensing wash solution to multipleplates. When the wash is performed by back-and-forth flow, the liquidhandling head can process only one plate (96 samples) at a time.

After the wash step, air is passed through the columns to remove anyorganic solvent remaining from the wash step. This can be accomplishedby depositing the pipette tip columns onto a drying station or vacuumblock and drawing air through the columns with a vacuum. A vacuum blockadaptor or drying station was custom built for this process and isdescribed in more detail below.

In certain embodiments, air is passed through the columns long enough toremove the organic solvent present in the wash solution, but not longenough to dry the columns completely. In other embodiments, the columnscan be dried completely. When the residual organic solvent is measured,it is in the range of less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2% or less than 1%.

In other embodiments, air is passed through the columns with positivepressure. Alternatively, it is possible to dry or remove the ethanol orother organic solvent after elution by methods such as speed-vac, airdrying, heating or applying a gas stream to the wells containing theeluted sample.

The elution of DNA from the columns can be accomplished with back andforth flow or unidirectional flow. Generally elution volumes are in therange of about 1-5 times the bed volume. When back-and-forth flow isused, air can be aspirated into the pipette tip column prior toaspirating the elution buffer. This air can be used after expulsion ofthe DNA to ensure complete expulsion of all the liquid in the column.

Generally, the elution buffer is aqueous and has a pH between 6 and 10.In some embodiments, the column is incubated with the elution buffer fora period of time. In these embodiments, the column and elution bufferare incubated for at least 1 minute, at least 2 minutes, at least 3minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes orat least 15 minutes. In other embodiments, the incubation step isomitted.

After the incubation step, the purified DNA is expelled from the pipettetip column. To ensure the maximum volume of purified DNA is recovered, ablow-out step can be performed by expelling the air aspirated asdescribed above.

When plasmid DNA is purified by the methods of the invention, generallythe concentration is at least 25 ng/μL. In some embodiments, theconcentration of purified plasmid DNA is at least 50 ng/uL, at least 75ng/uL, at least 100 ng/uL or greater. The A_(260/280) ratio of plasmidDNA purified by the methods of the invention is in the range of 1.8 to2.0. Most importantly, the plasmid DNA purified by these methods is highquality, substantially free of endotoxin and can be used for anydownstream application including sequencing, transfection andtransformation.

The entire process from cell harvest to eluted plasmid DNA generallytakes between 30 minutes and 1.5 hours. However in some embodiments, theentire procedure can be performed in the range of 40 to 80 minutes orbetween 45 and 75 minutes.

Plasmid Preparation Method Development

To develop a robust method for purifying plasmid DNA from an unclarifiedlysate, experiments were performed in which plasmids were purified fromE. coli cells. After the cells were grown, they are collected bycentrifugation and the growth medium is discarded. step for purifyingnucleic acids is cell lysis. Lysis can be carried out by a number ofmeans including the use of chemicals i.e., detergents or bymechanical/physical means, such as temperature or sonication.

Currently, the predominant commercially-available formats for plasmidpurification are spin columns, magnetic beads and vacuum plates. Inthese methods, cell debris is usually removed completely bycentrifugation after the lysis and neutralization steps to obtain a“clarified lysate” from which nucleic acids are purified (FIG. 5A). Theclarified lysate is free of all cell debris and flocculent. Completeremoval of cell debris to produce a clarified lysate is most oftenaccomplished by centrifugation but some vacuum plate methods utilizefiltration. These methods are undesirable because they aretime-consuming and difficult to fully automate, making it quitelaborious to purify nucleic acids from multiple samples simultaneously.Furthermore, when filtration is used, the filter is prone to clogging.Partial filtration is not subject to the same pitfalls.

Formats for plasmid preparation via vacuum include individual columnsand multi-well plates. Even after producing a clarified lysate, thesemethods are not well suited for automation. Some protocols recommendturning off the vacuum while adding reagents, which requires operatorinvolvement. Additionally, differences between samples can causedifferential column pressures between columns or wells within the plateso an operator is often needed to ensure the vacuum manifold seal ismaintained or that the liquid sample flow occurs evenly through all thewells of the plate. Since spin columns require a series ofcentrifugation steps, they are not amenable to automation withoutspecial equipment. Magnetic beads are expensive and require repeatedshake and aspiration steps, which makes their use difficult to automate.Magnetic beads or other bead suspension methods that do not first removethe cell debris are not reproducible and are difficult to automate.

An advantage of the instant invention is that plasmids can be purifiedin parallel, up to 96 samples at a time without operator involvement.With proper instrumentation, multiple plates of 96 samples can beprocessed simultaneously.

An experiment was performed to compare the viscosity of the unclarifiedlysate to that of a clarified lysate. An overnight culture of E. coliharboring a plasmid was subjected to centrifugation, resuspension, lysisand precipitation. A clarified lysate was made from half the mixture andthe viscosity was compared to the corresponding unclarified lysate. Foreach sample, the efflux time was measured using a size 400 Cannon-FenskeRoutine Viscometer. The efflux time is the time it takes for thesolution to travel between two points within a glass tube. The effluxtime for the unclarified lysate was almost twice as long as that for theclarified lysate (2.17 seconds vs. 1.18 seconds). The kinematicviscosity was calculated to be 2.6 centistokes for the unclarifiedlysate compared to 1.4 centistokes for the clarified lysate. In the somesamples including the more concentrated midiprep (described below), theviscosity of the unclarified lysate can be even higher. In certainembodiments, the viscosity can be in the range of 2 centistokes to 7centistokes, or in the range of 2.5 to 5 centistokes.

In U.S. patent application Ser. No. 12/767,659, a first approach toplasmid preparation from an unclarified lysate was described. Althoughthe invention described in U.S. patent application Ser. No. 12/767,659was an advance over other methods, the results obtained were stillinconsistent. Sometimes, the columns plugged with particulates containedin the unclarified lysate. In some cultures, the particulates seemed tobe greater in mass and all or most of the columns plugged. Even if theprocedure worked without incident at times, the recovered, purifiedvector performed well for sequencing but sometimes couldn't be usedeffectively for transfection or transformation. Another problem observedwas that the A₂₆₀ was artificially high at times, particularly when theplasmid was present in a low or medium copy number. After plasmidpurification, the concentration was measured by UV and also by asemi-quantitative measurement of the intensity of the plasmid band on anagarose gel. The comparison of these two methods suggested thatsomething present in the sample might be co-purified with the plasmid,causing the A₂₆₀ to be artificially high.

In the instant application, these problems were solved making the methodsignificantly more robust and reliable. Better sampling and purificationmethods were developed along with methods that allow scale-up in anautomated format. The quality and purity of the product was improvedmaking it useable for a greater variety of downstream applications.

To address the problem of random column plugging and increase thereproducibility of the method, we examined and developed an entirely newsampling procedure. It was discovered that the amount and type ofparticulate in unclarified lysate varied depending on a number ofparameters including medium, strain, replicon, growth time andconditions. It turned out that the distribution of the cell debrispresent in the sample differed dramatically between samples. Sometimesthe debris was distributed more or less throughout the sample, sometimesthe majority of the debris floated, but in other instances a portion ofthe cell debris sank. This variability seemed to be one reason themethod was not reproducible and that sometimes the columns plugged.Another reason seemed to be the amount of mass particulate variedtremendously from sample to sample. In some cases, the floating mass ofparticulate appeared to take up a large part, or even most of thesample.

It was desirable to use all the liquid in the unclarified lysate inorder to obtain the maximum amount of plasmid DNA. However, particulatemasses present in the lysate contained liquid that appeared entrainedand occluded. There did not appear to be active exchange of the occludedliquid with the other liquid in the sample.

Generally, in a suspension of particulates and liquid, the liquid canmove freely throughout the sample. But when masses or globs ofparticulate accumulate in a sample in a stable form, free movement ofthe liquid within the mass is halted. The mass of particulate is almostlike a large hydrated bead; there is no active transport of liquids butonly diffusion. The masses within the unclarified lysate looked globularand gel-like. It was speculated that plasmid contained in these globuleswould be unreachable unless the masses were broken up because activetransport of liquid in and out of the mass would be limited. In themethods described in U.S. patent application Ser. No. 12/767,659, theentire sample was passed through the pipette tip column. Passing thesemasses through the column broke up the masses and allowed capture of theplasmids. The only way to capture plasmid contained in the entiresample, including the sample within this occluded liquid, was to passthe entire sample through the column. This method worked because thepipette tip columns are designed to allow particulates to pass throughhowever, the columns clogged at times. It was desirable to develop amethod that reduced the amount of particulate in the sample while stillallowing automation.

Improved Sampling Method

A novel sampling method was developed to improve plasmid isolation fromthe unclarified lysate. First, the solutions were changed. In patentapplication Ser. No. 12/767,659, we used a lysis solution followed by aneutralization buffer comprised of a chaotropic salt, a salt and anacid. However, it was determined that it was more effective to use twosolutions sequentially. The lysis solution was first followed by asolution for neutralization (acid and salt) and then a second solutioncontaining the chaotropic reagent. When a solution containing salt andacid were added prior to the chaotropic salt solution, the A₂₆₀ moreaccurately matched the plasmid concentration obtained by the agarose gelband intensity. In addition, the amount of precipitate or cell debrisgenerated seemed to be more uniform.

However, this did not solve the reproducibility and plugging issue.There were still large amounts of particulate masses in the sample thatcontained entrained liquid. In some cases, these masses floated, whilein other cases, the masses precipitated. Some particulate remained insuspension of the sample but depending on the cell growth conditions andtime, the mass of cell debris appeared to make up about 20-50% (vol/vol)of the sample.

In commercially-available methods, the sample is centrifuged at thisstage and the supernatant (the clarified lysate) is used from plasmidcapture. Once the sample has been centrifuged, the liquid is very easyto process using spin columns or plates.

In the unclarified lysate used in the invention, it is likely that theactual solids content in the masses was only a very small portion of thesample. But having a substantial proportion of the sample entrained oroccluded within the floating or sinking masses seemed to be the majorissue. The liquid entrained within the mass of solid did not appear tobe available for capture unless there was active transport of the liquidto the resin in the column.

A second change made to the sampling procedure was that only a portionof the sample was aspirated and expelled. Instead of aspirating theentire unclarified lysate, only a portion was sampled. Quiteunexpectedly, it was determined that as little as 10% of the totalvolume could be repeatedly aspirated and expelled and the yield was notaffected provided the number of cycles of liquid traveling through thecolumn was increased. The term, “cycle” as used herein is defined as asingle aspirate/expel step.

Without being bound by theory, it is possible that the mass ofparticulate broke up and reformed with each expulsion of the liquid backinto the sample thus releasing or exchanging some of the entrainedliquid. It did not seem possible that diffusion of the plasmid from theoccluded liquid could occur because the distance to diffuse would beseveral millimeters and could even be more than a centimeter in somecases.

The experiments showed that sample volumes as low as 10% of the totalvolume in the well could be sampled and still get adequate recovery. Asmuch as 90% of the volume could be sampled while still eliminatingplugging of the column and get good recovery of the plasmid DNA.Preferably, between 10 and 90% of the sample volume can be sampled, morepreferably 20-80% of the volume can be sampled, more preferably 30-70%of the volume can be sampled, more preferably 40-60% of the volume canbe sampled, most preferably 35-50% of the unclarified lysate volume canbe sampled. These results were unexpected and surprising in light of thefact that the particulates were often globular and appeared to haveliquid sample entrained which had appeared to prevent capture of theplasmid within this liquid volume.

In some embodiments, the sampling procedure was modified to include theaddition of an aspirate and expel step prior to plasmid capture. Thisstep was performed while the pipette tip columns are attached to therobotic head. Air was drawn into the columns slowly and then the columnswere submerged in the sample and the air was slowly expelled through thecolumns into the unclarified lysate. The additional aspiration stepcaused the bulk of the particulates to float which more effectively keptthem farther away from the open lower end of the column during thesubsequent aspirate/expel cycles used for plasmid capture.

In certain embodiments, a carbonate, e.g. ammonium carbonate can beadded to the sample to make the flocculent float. In fact, any carbonatecompound can be used.

Improved Plasmid Quality

In U.S. patent application Ser. No. 12/767,659, removal of theinterstitial liquid from columns by vacuum or air pressure wasdescribed. Only a short duration of vacuum or air pressure is requiredto remove this bulk (interstitial) liquid: 0.1-1 minute or even between5 and 30 seconds depending on the force of the vacuum or air pressure.

In the methods described in U.S. patent application Ser. No. 12/767,659,bulk liquid was removed and the plasmid or nucleic acid was recoveredfrom the column by passing water or buffer through the column. Thequality of the plasmid was good and it was suitable for downstreamprocessing such as sequencing, mutation analysis, etc. However, it wasdiscovered that the plasmid recovered from this process could not beused successfully for transfection. Gels of the purified plasmid showedpure, concentrated plasmid yet, transfection frequency was very low.

The procedure described in U.S. patent application Ser. No. 12/767,659yielded a suitable quantity of plasmid DNA that performed well in DNAsequencing, however it was discovered that the baculovirus transfectionand bacterial transformation efficiency were both unexpectedly low.Initially, it was thought that the low transfection frequency was due tocontamination with protein, guanidinium or perhaps endotoxin. Endotoxinwas measured as described below and protein was measured by absorbanceat 280 nm. As a result, endotoxin and protein were ruled out ascontaminants. It was considered that there could be a nucleic acidcontaminant in the recovered plasmid such as genomic DNA or RNA.However, it wasn't possible to directly measure the genomic DNA or RNAcontamination.

Finally, it was suggested that a measurement should be performed ofresidual solvent in the recovered plasmid since the Wash buffercontained ethanol. The columns appeared to be free of solvent before theelution step and there was no indication that the recovered plasmidcontained any ethanol. The interstitial liquid in the column prior toelution appeared by visual inspection to be completely removed.

A Carl Zeiss single optic stand held refractometer was used to measurethe alcohol content in the purified plasmid. Aqueous standardscontaining known concentrations of ethanol were prepared and an analysisof the recovered plasmid was performed on several samples. Surprisingly,the samples of purified plasmid contained considerable amounts ofethanol, in the range of 5-15% (vol/vol). This result was surprisingbecause it was thought that ethanol would prevent efficient elution ofthe plasmid from the column. There is no alcohol in the elution solventin order to get efficient elution. The presence of ethanol was alsosurprising because a miniprep performed using a commercially-availablespin column method produced a final alcohol content in the recoveredplasmid in the 2-3% range. So clearly, something about the columns orthe method caused the residual ethanol to be present.

It was known that as the particle size of the resin used in the pipettetip columns was large. This was because the frit pore size of thecolumns had been increased to reduce plugging and therefore the particlesize of the resin was also increased so that it did not fall out of thecolumn. Without wishing to be bound by theory, it was known that theresin can contain pores to increase surface area and facilitate plasmidcapture. Unfortunately, the resin appeared to retain much more solventthan the spin columns, possibly due to its higher porosity and greatersurface area. In addition, the centrifugal force applied to spin columnsis probably quite efficient at vacating any liquid remaining in thecolumn. This retained solvent may have contributed to the higherpercentage of ethanol present in the eluted plasmid. Alternatively, thehigher percentage of alcohol obtained from the pipette tip columns andmethod could have been due to some other unknown phenomena.

Several different remedies were tested to solve the problem of residualorganic solvent in the purified plasmid. The first method evaluated wassimply to lift the columns out of the wash solution and pass air backand forth through the column with the robotic pipette head. Even thoughthe resin bed appeared to be equally wet at the beginning and end of theprocess, the amount of organic solvent in eluted plasmid decreased.While this method would likely work if the back and forth flow wasperformed with adequate number of cycles, it was not preferred becauseit added too much additional time to the method.

Other options to pass air through the columns were considered. Air couldbe forced through the columns by positive pressure however, this wouldrequire an additional apparatus be designed and built. Vacuum could beused, not only to remove bulk liquid, but as an additional stepimplemented to move air through the columns after the bulk liquid hadbeen removed from the interstitial space. A vacuum pump rated to pull 4cubic feet per minute through the pump at zero vacuum was used to pullair through columns under a number of different conditions.

These first experiments involved forcing air through set of 80 μL bedcolumns in a 96 well format and measuring the effect of total airthrough the system. After 1 minute and removal of the interstitialliquid, the total air pulled was measured to be 4 cubic feet. Themeasurement was performed by taking a venturi-type air flow meter andconnecting the meter to the vacuum in the reverse connection so that theair pulled through the meter was measured (rather than the normalmeasurement of air pushed through the meter). The initial experimentsshowed that the liquid was pulled through the column. After the initialliquid was removed, air appeared to be pulled through the columns.

The vacuum method was investigated by depositing the pipette tip columnsinto a vacuum station on the robot deck and passing air through thecolumns using vacuum. An oil vacuum pump (0.5 horsepower) was used topull a vacuum of 4 ft³/min through the columns. This use of vacuum isquite distinct from the traditional use of vacuum. Traditionally, vacuumis used to pull liquid solutions through plates or columns. After thesolution passes through the plate or column, the vacuum is turned offbecause the task has been accomplished. In the case of the instantinvention, the wash solution had already been passed through the columnsand the vacuum is used simple to draw air through the columns.

However, when measurements were performed with an air flow bubble meter(also connected in reverse) on individual wells, it was determined thatthe air flow after the interstitial liquid was removed from the columnwas not consistent from column to column. In fact, it was found that noair, or very little air was flowing through many of the columns whileother columns had significant air flow-through. Upon furtherinvestigation, it was determined that once the initial liquid had beenremoved from the columns, the vacuum seal formed for each column wasinconsistent. Analysis by refractive index of the elution solvent pulledthrough a number of columns indicated there was a correlation betweenthe quality of the vacuum seal and the amount of ethanol recovered withthe solvent. That is, those columns with a poor seal contained moreethanol while those columns having a good seal contained less. However,there was no difference in appearance of the individual columns. Theyall looked as though the interstitial liquid had been removed and theyall looked equally wet with surface liquid.

The next process tried was use of a 96-well aluminum heating block ovenand a forced air oven. The ovens were set to 37-42° C. After final washand expulsion of as much liquid as possible, the columns were placed inthe ovens for 10-30 minutes. Again, the columns appeared wet afterincubation in the ovens however, the ethanol concentration was reducedto as low as 5%. This result was encouraging however, the time requiredwas still longer than desired.

It was necessary to build a custom 96-well vacuum block. To test theeffectiveness of the vacuum block, it was necessary to build twoadditional air flow measurement apparatus. It was not possible tomeasure the air flow by seeing the liquid flow through the columns. Theair flow had to be measured directly. The first apparatus was a coverfor the vacuum block that was attached to and air gauge and used tomeasure air flow through the entire block. The air gauge (KingInstrument Company, Part No. 75201102C17) was actually used in reverse.That is, air was pulled through the top of the gauge rather than beingpushed through the bottom of the gauge as it was designed. Using thiscover, a reading greater than 0.4 cfm was achieved with the pump andblock being tested. Lifting the block from the vacuum manifold showedthat there was a good seal between the vacuum block and its manifoldbase.

After redesigning of the vacuum block, the air pulled through by vacuumafter the interstitial liquid was removed became more consistent betweencolumns. More ethanol was found when the air flow was slower, eventhough the columns appeared the same regardless of the airflow duration.It turned out, the vacuum generally pulled air through the column on anequal basis although the columns on the outside of the vacuum blockstill had higher flow than the center columns. Presumably this simpledesign permitted vacuum to pull the interstitial liquid through thecolumns, but once this was done, the vacuum applied to the columns wasinsufficient to apply uniform vacuum to all of the individual columns.

Several vacuum blocks were built before an adequate block design wasfound. The first block built had 96 positions on the top for the columnsand an open architecture on the bottom of the block. The air flowthrough the block seemed adequate. However, it was not possible to get atight seal when this block was tested with the cover. It seemed possiblethat while the total air flow may have been adequate, the air flowacross the individual columns could differ dramatically.

A second custom apparatus was built to test the vacuum throughindividual columns seated in the vacuum block. In this case a bubblemeter tube for measuring gas flow out of a packed bed gas chromatographwas modified to measure vacuum. A Wilmad LabGlass 10 mL gas flow bubblemeter was adapted to measure air flow through the individual columns. Aswith the other gauge, the vacuum was applied to the top of the metertube, leaving the tube fitting open that would normally have been theinlet from the gas chromatographic column. 96 columns were placed in theblock and the air flow through each column was measured. Using thisgauge, it was discovered that flow was not even between the columns. Tosolve this problem, the vacuum block was redesigned to have a gasketseal around each column.

The design of the column seal(s) proved to be difficult. The seal had tobe tight enough to seal all of the columns routinely and adequately. Butthe column had to be easily placed into the apparatus and it must bepossible to remove the columns from the block without the block beingpulled up along with the columns. The seal cannot be so tight as toprevent engagement of the columns by the robotic head. If the columnsseals were too tight, attempting to remove the columns from the blockcould result in the block being lifted with the columns. So the sealcould not be too tight. After several redesigns the block applied vacuumevenly through all the columns. Interestingly, it was not possible todetermine whether air flowed through a particular column or not byvisual inspection. Only the custom measurement tools could provide thisinformation.

The redesign of the vacuum block provided a tighter seal around eachcolumn while still allowing the columns to be removed from the block.After the redesign, experiments were performed to determine how muchairflow was needed to remove the residual ethanol from the column. Thelevel of vacuum and the vacuum duration were varied. In another set ofexperiments, the number of columns to which vacuum was applied wasvaried while keeping the vacuum level constant. In all experiments, thecolumn appeared dry by visual inspection before and after the vacuum wasapplied. The results showed that for 96 columns with an 80 μL bedvolume, a vacuum of 4 cubic feet per minute (CFM) applied for between 1to 20 minutes (above and beyond the vacuum needed for removal of theinterstitial liquid) was needed to lower the ethanol concentration ofthe eluent to 0-5%. This corresponds to an amount of 4-80 cubic feet ofair passed through 96 columns.

The next step involved testing the vacuum procedure for removal of theorganic solvent present in the wash solution. Liquid containing variousamounts of ethanol was cycled through the columns. The columns wereplaced in the vacuum block and vacuum was applied for varying amounts oftime. The columns were eluted with water, the eluant collected and therefractive index was measured for organic solvent concentration. Afterthe solvent removal step, the columns still appeared wet by visualinspection. To maintain the highest possible throughput, it wasdesirable to find the shortest possible vacuum duration that resulted inpurified plasmid having acceptably low alcohol content. Although thesolvent drying step is an additional step to the process, if a verystrong vacuum is used, the columns can be dried more quickly withoutsacrificing throughput.

Depending on the vacuum applied and the air flow through the individualcolumns, the “drying time” can be between 30 seconds and 20 minutes, butpreferably between 2 and 5 minutes. Drying time is defined as the timethat vacuum or air flow is applied after the removal of the bulk liquid(which can also be done by vacuum or air). Based on these experiments, avacuum duration was determined for which the eluent contained anacceptable amount of ethanol. Preferably, the percentage of ethanol inthe purified plasmid is less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2% or less than 1%. Less than 5% solvent was preferred and lessthan 3% was most preferred.

In other embodiments with longer drying conditions, it is possible todry the columns completely prior to elution of the purified plasmid.However, good results were obtained when the solvent was substantiallyremoved and the columns were not dried completely after the final washand prior to elution.

Implementation of the solvent removal step affected the reproducibilityof the elution step. For example, when 80 μL bed silica columns weresubjected to vacuum to remove the organic solvent present in the wash,130 μL of water was used for the elution step and only 80-90 μL ofliquid was collected. This result indicated that a significant portionof the water was trapped in the dead volume of the pipette tip column.

To help mitigate this problem, the elution step can be modified. Anadditional step can be added at the end of the procedure to maximize theelution volume. Air can be taken up by the robotic pipetting head afterthe solvent removal step and prior to engagement of the columns forelution. This added air is then expelled after expulsion of the purifiedplasmid to get as much liquid as possible out of the column.

Yield can also be increased by incubating the elution solution on thecolumn prior to expulsion. As an example, the elution solvent can beaspirated and incubated on the column for 5 minutes prior to expulsion.

Partial Filtration

After lysis and precipitation, the sample contains a flocculentcomprised of reagents and cell debris. It can be advantageous to removesome of the flocculent by partial filtration. Although the columns cantolerate particulates in the sample, the sample can be processed morerapidly and more completely if cell debris is at least partiallyremoved. After the partial filtration step, the lysate still containssome particulates. Since the filtrate still contains some cell debris,it is not considered a clarified lysate. Instead, the filtrate is called“a partially-clarified lysate.”

A filter with very large pores can be used to remove some of theflocculent. A large-pore filter is defined herein as a filter having apore size larger than 25 μm. In certain embodiments, the large-porefilter has a pore size greater than 30 μm, 35 μm, 40 μm, 45 μm or 50 μm.Generally, the pore size for a large pore filter is in the range of 50micron-3000 micron. For partial filtration, a large-pore filter is used.The pore size of the filter is generally in the range of 200 to 2000 μm.In certain embodiments, the pore size is in the range of 500 to 1500 μm,600 μm to 1000 μm or 700 μm to 900 μm.

Partial filtration performed with a large-pore filter quite unorthodox.While commercially-available filters generally have a pore size of 25micron or less, the pores of the large-pore filter are much larger. Inthe large-pore filter, the pores are larger than some of the particulatematerial found in the sample. As a consequence, some particulatematerial goes through the filter. In contrast to conventional filters,the large-pore filter is not designed to filter all particulate; just amajority of the particulate material.

In addition, the large-pore filter is designed to operate under gravity,low pressure or vacuum, even in automated embodiments. The large-porefilter can operate at low pressures. For example, the filter can operateat less than 20 psi, less than 15 psi, less than 10 psi, less than 5psi, less than 4 psi, less than 3 psi, less than 2 psi, or less than 1psi.

For automated embodiments, it is unusual to have a filtration apparatusoperated using gravity because the filtration rate is unknown. Theamount of particulate is sample-dependent and therefore unpredictable.Generally, automated procedures require pre-programmed times andvolumes. Similarly, it is unusual to have in automated filtrationprocess operated using low pressure or vacuum.

There are a number of different strategies for automated the partialfiltration process. In general, the bacterial culture is centrifuged andthe medium is decanted. The pellet is then resuspended in a buffer and alysis solution is added followed by a precipitation solution asdescribed above in the section entitled, Purification of DNA from E.coli. These steps can be performed in a tube, centrifuge bottle oranother type of container. After the precipitation step, the sample ispartially filtered. In certain embodiments, the sample can betransferred to the partial filtration apparatus in an automated fashion.In other embodiments, the container, tube or centrifuge bottle harboringthe precipitated sample can be placed above the partial filtrationapparatus and pierced in such a way that the sample flows into thefilter.

Partial filtration using gravity flow can be performed in 15 minutes orless. In some embodiments, gravity-flow partial filtration can beperformed in 10 minutes or less, 5 minutes or less, 2 minutes or less,90 seconds or less, 1 minute or less or 30 seconds or less.

The filter can be comprised of any material or combination of materials.Nonlimiting examples include plastic, metal, nylon, glass, cloth as wellas any of the materials listed below in the section on frits. In someembodiments, the filter is a coarse or porous cloth. In theseembodiments, several layers of cloth can be used. Smaller particles passeasily through the cloth filter, but much of the particulate isretained.

The filter pores can have any geometric or irregular shape. For example,the pores can be round, oval, square, rectangular or polygonal. Forpores that are not round, pore size is defined herein as the average ofthe longest distance across the pores.

Partial filtration generally removes between 50% and 99% of theflocculent or cell debris. Conversely, 1% to 50% of the flocculent/celldebris can pass through the large-pore filter. Since the density of theflocculent/cell debris is similar to that of water, 50% of theflocculent/cell debris can be considered either 50% (wt/vol) or 50%(vol/vol).

In certain embodiments, partial filtration can remove between 60% and99%, between 60% and 98%, between 65% and 97%, between 70% and 96% orbetween 80% and 95% of the flocculent. In other embodiments, partialfiltration can remove between 50% and 97%, between 50% and 96%, between50% and 95%, between 60% and 90% or between 70% and 90% of theflocculent. In terms of the percentage of flocculent that can passthrough the filter, that can be between 1% and 40%, between 2% and 40%,between 3% and 35%, between 4% and 30%, or between 5% and 20%. Thepercentage of the flocculent that passes through the filter can becalculated by determining the volume of the flocculent and the totalvolume of the filtrate.

In some embodiments, gravity flow is used to partially filterparticulate. In other embodiments, pressure or vacuum is used to forceliquid through the filter.

The partial filtration step can be performed in an automated fashion orthe automated procedure can begin after the partial filtration step. Insome embodiments, automation begins early in the plasmid purificationprocess, e.g. at the cell pellet resuspension step. In otherembodiments, the automated process can begin after partial filtration,particularly for larger preps such as maxi, mega and giga.

In some embodiments, the filter is vertical while in other embodimentsfilter is horizontal. The area of the filter may be on the order of 3cm², 5 cm², 10 cm², 20 cm², 30 cm², 40 cm², 50 cm², 60 cm², 80 cm², 100cm², 125 cm², 150 cm², 200 cm², 400 cm², 600 cm², 800 cm² or 1000 cm².In some embodiments, a single filter is used to remove particulate fromthe plasmid sample. In some embodiments, two or more filters operated inseries may be used to remove particulate from the plasmid sample. Whentwo or more filters are used in series, it is possible to remove most orall of the particulate from the sample.

FIG. 6 shows a cut-away view of one embodiment of a partial filtrationapparatus. The precipitated lysate 22 is deposited on large-pore filter33. Filtrate 44 is the partially clarified lysate. Following thefiltration process, the filtrate can be removed through open space 55.

Endotoxin Removal

Endotoxin is an abundant lipopolysaccharide present in the cell wall ofgram-negative bacteria such as E. coli. It is undesirable to haveendotoxin molecules in the purified plasmid DNA because it can stimulatethe mammalian immune system, decreasing transfection efficiency.

Several different methods can be used to remove endotoxins from nucleicacid samples. For purification procedures that use ethanolprecipitation, endotoxin is commonly removed during the ethanolprecipitation step. For procedures that don't employ ethanolprecipitation, a dedicated resin can be used for endotoxin removal (seeU.S. Pat. Nos. 6,194,562 and 6,942,802). Another method involves aborate wash (see U.S. Pat. No. 7,935,505). Yet another technique forendotoxin removal involves enzymatic destruction as described inW02007115046.

In the instant invention, none of the methods described above are used.Nucleic acids are captured on the column, the column is washed and thenucleic acids are eluted. The wash solution does not contain borate.

An endotoxin test was carried out to examine concentration of endotoxinobtained from miniprep spin columns versus the miniprep pipette tipcolumns and methods of the invention. Plasmid DNA was purified intriplicate from E. coli pellets as described above. Additionally,plasmid DNA was purified in duplicate using a spin column method (QiagenCat. #27104) according to the manufacturer's protocol. The plasmid DNAswere tested using a ToxinSensor Chromogenic LAL Endotoxin Assay Kit fromGenscript (Cat. L00350C). Using the supplied endotoxin controls, astandard curve was generated, and the amount of endotoxin in the plasmidDNA preparation was quantified. In all cases, endotoxin was measured tobe less than 0.01 endotoxin units per microgram of DNA, well within theacceptable levels for transfection experiments (Table 1).

TABLE 1 Colorimetric Endotoxin Assay of Purified Plasmid DNA Vol.[Endotoxin] [plasmid] Tested [Endotoxin] Plasmid [Endotoxin] A₅₄₅(EU/mL) (ng/μL) (μL) (EU/100 μL) (μg) (EU/μg) Spin column 1 0.486 0.17183 100 0.017 18.3 0.0009 Spin column 2 0.575 0.20 183 100 0.020 18.30.0011 Pipette tip col. 1 0.526 0.19 76 100 0.019 7.6 0.0025 Pipette tipcol. 2 0.477 0.17 76 100 0.017 7.6 0.0022 Pipette tip col. 3 0.472 0.1776 100 0.017 7.6 0.0022

Each bacterial cell contains approximately 2 million endotoxinmolecules. For the larger scale preparations (maxi, mega and giga)endotoxin removal is more difficult. Because of the large volumes ofbacterial culture processed (and the larger number of cells), the columnresin is loaded quite heavily relative to the loading of the smallercolumns. That is, the amount of endotoxin relative to the capacity ofsolid phase is greater. As a consequence, the final concentration ofendotoxin in the purified DNA is somewhat higher. In these largerpreparations, it is more difficult to obtain a concentration of 0.01 orless endotoxin units per microgram of DNA. A Maxiprep experiment yieldedendotoxin concentrations of between 11 and 16 EU/μg.

Although the endotoxin concentrations were higher with the largerplasmid preparations, transfection frequency was still high. As aconsequence, the term, “substantially endotoxin-free” is defined hereinas an endotoxin concentration of less than 100 EU/μg.

Scale-Up to High-Throughput Midiprep

Another significant point of novelty of the instant invention is in thearea of scale-up. Plasmid purification protocols are typically called“miniprep”, “midiprep” or “maxiprep” based on their scale and yield.Although these plasmid purification protocols are known in the art, anautomated system for performing 96 midiprep at a time has not beendescribed previously. In the instant application, scale-up to ahigh-throughput midiprep was achieved.

For the purpose of this invention, an automated miniprep method isdefined as a method in which the amount of plasmid or nucleic acidrecovered is in the range of up to 30 μg as shown in Table 2. A scale-upto midiprep was achieved so that 96 samples were processedsimultaneously with a yield of plasmid DNA in the range of 20 to 200 μg.For a midiprep, the yield can be in the range having a lower limit of 20μg, 25 μg or 30 μg and an upper limit of 50 μg, 60 μg, 80 μg, 100 μg,120 μg, 140 μg, 160 μg, 180 μg or 200 μg. Therefore, a midiprepautomated approach is defined as a method where the amount of plasmid ornucleic acid recovered is in the range of 20-200 μg per column. Thelysate used for this method can be unclarified, clarified orpartially-clarified.

TABLE 2 Comparison of miniprep in midiprep Mini Midi Growth 1-5 ml 2-30mL volumes Resuspension 150 μL 200 μL buffer Lysis buffer 180 μL 510 μLPrecipitation 210 μL 300 μL (PB1) buffer 530 μL (PB2) Column 1 mL 1 mLvolume Bed volume 10-120 μL 100-500 μL Yield up to 30 μg 20-200 μg

Scale-up to an automated, high-throughput midiprep cannot be consideredcase of optimization through routine experimentation or automating amanual procedure. Development of this parallel, automated midiprepprocedure required a number of additional technical obstacles to beovercome. It was not possible to simply scale up the bed volume andreagent volumes used in the miniprep because of the volume constraintsimposed by the use of 1-mL or 1.2-mL pipette tips used with automation.

To create an automated, high-throughput midiprep procedure, it was notpossible to simply scale up the column bed volume and all the solutionvolumes proportionally. The midiprep plasmid purification requires alarger amount of resin in each column when compared to the miniprepcolumns. It was challenging to increase the bed volume while stillleaving sufficient space for the solutions. The volumes of the samplesand the associated solutions used to process the samples increaseddramatically. To make the invention compatible with commercial liquidhanding systems and commercially-available pipette tips, a 1 mL pipettetip body was used. As a consequence, the column bed, sample andsolutions were limited to one mL.

Spin columns are not faced with this problem for scale-up. For example,a commercially-available miniprep spin column has bed dimensions of 7.0mm diameter and 2.05 mm height giving a bed volume of 79 mm³. When thebed material is scaled up to midiprep size, the bed dimensions increaseto 13.9 mm diameter and 11.2 mm height giving a bed volume of 1700 mm³.This is more than a 20 fold increase in bed size.

With the pipette tip format, it wasn't possible to scale up the columnbed volume 20 fold. The bed volume for the miniprep is 10-120 μL.Certainly, the bed size could be decreased if a lower recovery ofplasmid is desired. But, if a larger yield of plasmid is required, it isnot possible to increase the bed volume 20 fold as is the case with spincolumns. If the pipette tip column bed were increased 20 fold, it would2.4 mL, a volume too large for a one mL pipette tip. The pipette tipscannot hold enough medium for a 20-fold scale-up.

At the outset, it appeared to be impossible to scale the automatedmethod enough to obtain 20-200 μg of purified plasmid because of volumeconstraints for the solutions within the columns. Incommercially-available spin or gravity-flow columns, the volumes of thesolutions are increased 15 to 20-fold when scaling up from miniprep tomidiprep. Clearly, this was an added difficultly when scaling anautomated method performed in a 96-well format. If the resuspension,lysis and precipitation buffers were scaled up 20 fold, the total volumewould be over 10 mL. Two-mL deep well plates are the most common sizefor the 96-well format. Four-mL deep-well plates exist however they arenot readily available. Even with 4-mL plates, multiple wells would haveto be used to contain the unclarified lysate.

Growth volumes can be adjusted depending on parameters such as therichness of the growth medium and the copy number and size of thevector. In one embodiment, cells for mid5prep are grown in a flask ortube. For example, 30 mL of overnight culture is processed by a singlemidiprep column. In another embodiment the starting culture is between5-15 ml which produced approximately 50 μg of purified plasmid. With lowdensity cultures or low copy number vectors, it may be desirable toprocess 30 mL of culture to get recoveries of greater than 50 μg ofplasmid.

In some embodiments, growth for midiprep can be performed in multi-wellplates. For instance, cells can be grown in 6-, 12-, 24-, 48- or even96-well plates. When 96 midiprep columns are used and growth isperformed in plates having fewer than 96 wells, multiple plates can beused for growth, (e.g., four, 24-well plates can be used to grow cellsfor 96 midiprep columns.) In these embodiments, the consolidation frome.g., 24-well plates to 96 columns can be performed with a liquidhanding system. Alternatively, consolidation can be performed with amulti-channel or even a single-channel pipette.

Consider for example, cell growth in four 24-well plates for 96 midiprepcolumns. Consolidation from the 24-well plates to 96 columns can beperformed at varying stages during processing. In one embodiment, cellscan be transferred directly from the 24-well plates into one or more96-well plates. In a second embodiment, the 24-well growth plates can becentrifuged and the cell pellet can be resuspended by gentle mixing witha liquid handling robot as described previously. In this embodiment, theresuspension buffer volume can be chosen to yield the desired volume ofresuspended cells. That is, a small volume of resuspension buffer can beused to produce a highly-concentrated cell suspension.

One of the first problems tackled was the size of the column bed. It waspreferred that the bed size not be too large because of limited chamberspace above the bed. In certain embodiments, the bed volume is less thanhalf the volume of the pipette tip in which the column is made. In theseembodiments the bed volume can be less than ⅓ the volume of the pipettetip or less than ¼ of the volume. It is desirable to have considerablespace above the bed so that relatively large liquid aliquots can beprocessed by back and forth flow.

It is also possible to use a bed volume that is greater than half thevolume of the pipette tip in which the column is made. This is notpreferred however for several reasons. First, a larger bed would giverise to higher resin costs. Second, a larger bed would result in alarger volume of eluted plasmid which could require furtherconcentration, e.g., by ethanol precipitation. Third, because the resinwould take up as significant portion of the column volume, it would benecessary to process smaller liquid aliquots during the capture and washsteps.

In one experiment, a 300 μL resin bed in a 1 mL pipette tip was tested.This bed height was 3.75 times higher than the miniprep columns (80 μLresin bed) described herein. In another experiment, the column bedvolume was 400 μL. Experiments were performed in which enough celllysate was passed through the column to load the columns to capacity.Surprisingly and unexpectedly, it was discovered that the resin did haveenough capacity to recover up 100 μg of plasmid. Without wishing to bebound by theory, the significant increase in plasmid yield may have beendue to the porous nature of the packing material. Nevertheless theresults were unexpected. The column bed size for midiprep recovery of20-200 μg nucleic acid recovery ranged from 85-800 μL, 200-500 μL or300-400 μL. In certain embodiments, the bed volume for a midiprep was inthe range of 100 μL to 500 μL.

The bed size can also be defined by the percentage of the pipette tipcolumn taken up by the bed. For example, an 85 μL bed in a 1.2-mLpipette tip takes up approximately 7% of the volume available in thetip. Therefore, the bed size for a midiprep can take up at least 7%, atleast 8%, at least 12%, at least 16%, at least 20%, at least 25%, atleast 29% or at least 33%, of the volume available in the tip.

Although it is the most economical to manufacture the columns fromcommercially-available pipette tips, it is also possible to make columnsthat can engage a liquid handler but are cylindrical in shape, or evenanother shape. In these embodiments, the resin can take up a smallerpercentage of the tip.

Next, the volume constraints of the resuspension buffer, the lysisbuffer, the precipitation buffer and the entire sample were examined. Asmaller volume of resuspension buffer could be used with the consequencethat the cell suspension would be more concentrated. A more concentratedcell suspension would give rise to a more concentrated lysate. Since thelysate is unclarified in some embodiments of the invention, a moreconcentrated lysate has more particulates, more cell debris and moregenomic DNA per unit volume, making it more difficult to process.

Alternatively, a larger volume of resuspension buffer could be used andthe sample could be captured from a number of wells, perhaps up to 4 ormore. However, the larger volumes are more difficult to work with andwould require additional disposables and expense. In one embodiment, themidiprep procedure employs 4 mL resuspension buffer, 4 mL lysis buffer,and 6 mL of precipitation buffer, making the total volume 14 mL. Avolume of 14 mL would require 8 wells of a 96-well deep-well block. Sowhile this embodiment would be possible to automate, it is not optimal.

In order to solve this issue, several smaller resuspension, lysis andprecipitation buffer volumes were tested to reduce the total volume weneed to process the midi sample:

-   1. 300 μL Resuspension buffer, 300 μL Lysis buffer, 410 μL    Precipitation buffer: total=1010 μL-   2. 500 μL Resuspension buffer, 500 μL Lysis buffer, 700 μL    Precipitation buffer: total=1700 μL-   3. 1 mL Resuspension buffer, 1 mL Lysis buffer, 1.4 mL Precipitation    buffer; total=3.4 mL-   4. 2 mL Resuspension buffer, 2 mL Lysis buffer, 2.8 mL Precipitation    buffer: total=6.8 mL

These volumes may be adjusted to produce more concentrated reagents.However, this may produce more particulate or the salts and buffersmaking up the reagents may become insoluble at the concentrationsrequired by the process. A range of reagent concentrations up to, andincluding those concentrations listed in Table 3 can be used in theautomated midiprep procedure.

In some embodiments, more concentrated neutralization/precipitationreagents can be used by performing the neutralization in two steps. Thatis, the guanidine hydrochloride could be added prior to the potassiumacetate or vice versa. In certain embodiments, these reagentconcentrations

TABLE 3 Midiprep buffers Buffer Name Content Resuspension 1M Tris-HCl pH8.0, 1M EDTA, 4 mg/mL RNase A buffer Lysis buffer 6M NaOH, 10% SDSPrecipitation, 10M guanidine hydrochloride buffer 5M Potassium acetatepH 4.5 Wash buffer 1M TRIS-HCl pH 7.5, and up to 100% Ethanol

In some embodiments, the ratio of Resuspension buffer to Lysis buffer toPrecipitation buffer is considered. This ratio can be 1:1:1.2. Incertain embodiments, less Resuspension buffer is used in order tominimize the total volume. That is, the ratio of Resuspension buffer toLysis buffer can be 1:1 or it can be less. For example, 150 μl ofResuspension buffer can be used with 500 μl Lysis buffer. When thissmall volume of resuspension buffer is used, the buffer can be 10-foldmore concentrated. Alternatively, 300 μl or 500 μl Resuspension buffercan be used with 500 μl Lysis buffer.

In certain embodiments, the resuspended cells can be transferred to a96-well plate for further processing. However, it is also possible tocontinue processing in the 24-well format. Cell lysis and precipitationcan be performed in the 24-well format, and the aqueous portion of theresulting unclarified lysate can be processed on a midiprep column.

In many embodiments, the plasmid is captured by repeated aspiration andexpulsion through the open lower end of the mid-prep column. In otherembodiments, the sample can be applied to the open upper end of the midicolumn and allowed to pass through the column by vacuum or gravity flow.

When capture is performed by repeated aspiration and expulsion, partialsampling of the unclarified lysate can be used as described above. Inone embodiment, partial sampling can be performed in several aliquots.After each aliquot is processed, it can be expelled to waste.

As with the miniprep, the wash and elution steps can be done withaspiration and expulsion or they can be done by using by gravity flow.When aspiration and expulsion are used, the buffers can be delivered ina top-down fashion (from above) or a bottom-up fashion (from a wellbelow the column).

The 96-well automated method can be performed with clarified,partially-clarified or unclarified lysates.

Scale-Up to Automated Maxi, Mega and Gigaprep

In another aspect of the invention, it is possible to automate largeryield plasmid preparations. To do this, it is necessary to scale up thebacterial culture volume, the column size, the column bed volume and thebuffer volumes respectively. Example volumes and yields are shown inTable 4. In Table 4, a gigaprep is comprised of two, megapreps. The twomegapreps can be performed separately and combined after column elution.

Similar to the midiprep, the column bed size can be characterized by thepercentage of the pipette tip column taken up by the bed. For the maxi,mega and gigaprep, the percentage of the column that is occupied by thesolid phase is in the range 4% to 55%.

In these embodiments, bacterial cultures can be grown in any of themedia described above. In certain embodiments, cultures are grown in therich medium such as Terrific Broth. A 20 mL pipette tip column can beused for the maxi, mega and gigapreps. Because of the larger columnsize, it is not possible to perform 96 preparations simultaneously.Nevertheless, it is possible to automate the procedure. In someembodiments, the preparations may be performed in parallel for between 2and 24 samples or more, depending on the scale.

TABLE 4 Comparison of maxiprep, megaprep and gigaprep Maxi Mega GigaGrowth 30-500 mL 500-1500 mL 2 L-3 L volumes Resuspension 30-60 mL100-300 mL 200-400 mL buffer Lysis buffer 40-70 mL 150-350 mL 250-500 mLPrecipitation 45-80 mL 150-350 mL 250-500 mL buffer Column 20 mL 20 mL 2× 20 mL volume Bed volume 1.5-5 mL 5-10 mL 5-10 mL Yield 0.5-1.5 mg 1-4mg 3-15 mg

Because of the larger column size, the throughput is lower than that ofmidiprep. However the system is largely automated nevertheless.

The procedure can be performed as follows.

-   1. Grow culture overnight.-   2. Transfer bacterial culture into centrifuge tubes and centrifuge    to pellet cells. Decant excess medium.-   3. Add Resuspension Buffer to tube with pelleted cells. Resuspend    completely by vortexing or pipetting.-   4. Add Lysis Buffer to resuspended cells. Invert or pipette several    times slowly.-   5. Add Precipitation Buffer to lysed cells. Invert or pipette    several times.-   6. Optionally, transfer total contents of tubes to a partial    filtration apparatus.-   7. Transfer the partially-clarified lysates to 50 mL conical tubes.-   8. Capture with a series of aspirate/expel cycles in each 50 mL    conical tube.-   9. Transfer the columns to the drying station.-   10. Add the Wash Buffer to columns.-   11. Add Elution Buffer to columns.

An instrument can perform most or all steps of the purification processin an automated fashion using computer or automatic control. In general,the instrument performs timed events in a predetermined sequence. Onceinitiated, the timing and sequence of events cannot be changed. Thismeans the process must be robust and versatile enough to be able toprocess samples that vary in composition and size.

Automation removes the drudgery of performing operations manually.However, automation is generally restrictive because there is noopportunity to change or adapt method to problems that may arise inoperation. For example, in manual plasmid preparation methods that usefiltration, the timing of the various steps is subject to changedepending on the amount of cell debris a particular sample. Because thelength of time necessary to achieve complete filtering may vary, it isdifficult to automate this step.

Hardware and firmware were combined to create an instrument capable ofautomated plasmid preparation at the maxiprep, megaprep and gigaprepscales. Columns, reagents, buffers, filters and containers are arrangedon a deck of an instrument that has x, y and z motion. Columns and othercomponents can be moved in the x, y and z directions. In someembodiments of the maxiprep, megaprep and gigaprep, the column bedvolume ranges from 0.5 mL up to 15 mL. However, the size and shape ofthe upper end of the column remains consistent so that majormodification of the instrument is needed to engage columns of varyingsizes.

Solutions are buffers on the instrument deck can be contained in anyvessel. In certain embodiments, solutions are held in conical tubes e.g.25-100 mL). Additionally, the instrument can accommodate larger bufferreservoirs (e.g. up to 2 L).

In some embodiments of the invention, the automated steps includefiltration, capture, wash and recovery of the plasmid. These steps maytake between 15 minutes and 120 minutes depending on ease of capture andthe number and extent washing needed. In other embodiments, one or moreof the operations covering cell resuspension, lysis left andprecipitation are included in the automation steps.

Samples are precipitated and can then be applied to a filter to removethe cell lysate particulate. In certain embodiments, the precipitatedsample is partially filtered using a large-pore filter while in otherembodiments, the particulate can be filtered completely. In still otherembodiments, and unclarified lysate can be used. in certain embodiments,a clarified lysate can be produced by centrifugation.

The filtrate is then applied to the column to capture the plasmid. Afterthe capture step, column is washed with one or more buffers to removeproteins and other material not of interest from the column. The columnmay F vashed several times with mt buffers. After the plasmid ispurified on the column, air can be passed through the column to dry thecolumn. After washing, the plasmid is eluted to recover the plasmid. Theelution may be performed with buffer or water. The plasmid can berecovered in a vial or tube.

In some embodiments of the instrument, between 1 and 24 columns can beautomatically processed in parallel. In other embodiments of theinstrument it is possible to process between 2 and 12 columns or between2 and 6 columns in an automated fashion.

In some embodiments, the procedure is automated from step 6 while inother embodiments, automation begins at step seven 7. In someembodiments, the procedure is automated from step 7. In alternativeembodiments, the procedure can be automated from the addition of theResuspension Buffer in step 3. In these embodiments, steps 3, 4 and 5can be performed using a series of pre-programmed aspirate/expel orstirring steps. When partial filtration is performed, the partialfiltration apparatus can be placed on the instrument deck.

As with the miniprep, the wash and elution steps can be done withaspiration and expulsion or they can be done by using by gravity orvacuum flow. The buffers can be delivered in a top-down fashion (fromabove) or a bottom-up fashion (from a well below the column).

The invention described herein includes an instrument capable oflarge-scale automated plasmid preparation. When a process is automated,it becomes less flexible and more limited because programmed events areperformed in a predetermined inflexible manner. However, the instrumentdescribed herein is capable of three different scales of plasmidpreparation: maxiprep, megaprep and gigaprep.

It was quite challenging to design an instrument capable of processingthree different scale preparations. The computer control was difficultto implement because the timing of each step must be predictable andreproducible. Timed events controlled by computer software and firmwaremust be determined. If timing for the various steps of the processes areunpredictable or uncontrollable, it is challenging to create anautomatic process control in which a sequence of predetermined times areused to perform the progression of events.

Failure of an automated method can occur for a number of reasons. Forexample, failure can be due to the sheer number of cells that requireprocessing as the method is scaled up. Indeed, as the scale of a plasmidprep is increased, the amount of cell debris or flocculent that must beprocessed also increases, which in turn increases the problem ofcomputer timing. Increasing the amount of cells in the sample increasesthe flocculent that must be filtered before the plasmid is captured. Butthere are limitations on the material that can be filtered, especiallyin an automated instrument. In addition, an instrument that performsdifferent scales of plasmid preparation must accommodate a wide range ofcell flocculent mass amount. The instrument must be flexible andpredictable to operate under conditions in which the amount of cellflocculent changes dramatically.

Currently, there is only one commercially-available instrument on themarket that performs automated plasmid maxipreps, the BenchPro 2100Plasmid Purification System (ThermoFisher Scientific). The instrumentdescribed herein has the numerous advantages over the BenchPro 2100.

The instrument of the invention is able to recover plasmids at threedifferent scales without modification of the instrument for manipulatingand using columns, buffer containers, filters and recovery vials. Thisis remarkable because the range of the number of cells that can beprocessed spans a range greater than 100.

This flexibility is accomplished using a uniform column size and columnengagement circumference for maxiprep, megaprep and gigaprep. The columnengagement circumference is defined herein as the distance around thecolumn that seals the column to the instrument. The seal may be topressure or vacuum to help process liquids through the column. Thecircumference may be any shape including circle, square, rectangle,polygonal, etc.

In some embodiments, the column body is the volume of 20 mL but thecolumn bed volume can vary depending on the scale of the plasmidpreparation. The instrument can engage columns of different bed sizeswithout modification of the instrument or the head used to engage thecolumns. The engagement seals the vacuum or pressure needed to forceliquid flow through the column. For 20 mL columns, the engagementcircumference is 6.3 cm. However, the instrument may operate usingdifferent size columns. Engagements circumferences can be in the rangeof 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50 or 60 cm. Theengagement diameter is related to the column diameter.

The BenchPro 2100 is limited to maxiprep scale. In contrast, theinstrument of the instant invention is more flexible and can performplasmid preparations at three different scales: maxiprep, megaprep andgigaprep.

There is a limit to the number of cells that may be processed in theBenchPro 2100 instrument. The BenchPro 2100 can only process a total of1.3×10¹¹ cells. In contrast, the instrument described herein can processmany more cells.

-   -   Maxiprep: 5×10¹²-5×10¹³    -   Megaprep: 1.5×10¹³-1.5×10¹⁴    -   Gigaprep: 3×10¹³-3×10¹⁴

However, the methods and devices described herein are not limited to thecell ranges listed above. It is also possible to process fewer cells. Infact, it is possible to process between 1.3×10¹¹ and 5×10 ¹² cells. Bythe same token, it is possible to prepare plasmid DNA from fewer than1.3×10¹¹ cells.

The BenchPro 2100 instrument can process a maximum bacterial culturevolume of only 125 mL grown in LB. In the instant invention, much largerbacterial culture volumes can be processed. Although gigaprep is usuallylimited to a maximum volume of 3 L bacterial culture, volumes of up to 5L can be processed in some embodiments. When 5 L of bacterial culture isused, up to 7.5×10¹⁴ cells can be processed. Furthermore, in the instantinvention, any medium may be used to grow cells prior to plasmidpreparation. This includes media much richer than LB such as TerrificBroth, SOB, SOC, 2×YT or Agencourt Ale.

The BenchPro 2100 instrument is comprised of several filters that areused at different stages of the plasmid preparation method. The surfacearea of these filters may limit the capacity of the BenchPro 2100. Whentoo many cells are present, it is possible to plug one or more of thefilters, preventing successful recovery of the plasmid. On the otherhand, the instrument described herein can operate with only a singlelarge-pore filter. Since the filter has large pores and it is used toperform a partial filtration step, it is not prone to clogging. Also,the same filter dimensions may be used for all three scales of sampleprep: megaprep, maxiprep and gigaprep scale.

Another limitation found in the BenchPro 2100 is the size of its buffercontainers. They are limited to 50 mL. In contrast, the instrumentdescribed above utilizes buffer containers up to 2 L. Larger containerscan be used if necessary.

In some embodiments, it is possible to scale up to a teraprep. In theseembodiments, the growth volume is larger than 3 L, on the order of 8-12L, columns would be 50-100 mL, bed volumes would be 20-40 mL and yelledwould be 12-60 mg.

The Columns

In the subject invention, a bed of medium is contained in a column,wherein the bed is held in place with a bottom frit. In someembodiments, the columns are additionally comprised of a top frit.Non-limiting examples of suitable columns, particularly low dead volumecolumns are presented in U.S. Pat. No. 7,482,169. It is to be understoodthat the subject invention is not limited to the use of low dead volumecolumns. The columns may be configured into plates or racks or usedindividually.

Typically, the column is comprised of a column body having an open upperend, an open lower end, and an open channel between the upper and lowerends of the column body; a bottom frit extending across the open lowerend and a bed of medium positioned inside the open channel above thebottom frit.

In certain embodiments, the column body is comprised of a pipette tip.FIG. 1 depicts an embodiment of pipette tip column construction.Disposable pipette tip 160 is cut at the lower end and frit 174 iswelded to the lower end of the tip body. A silica resin 184 wastransferred into the tip. In certain embodiments, upper frit 198 isplaced above the resin, e.g., using a friction fit. Pipette tip 190 canengage the liquid handling system used to process the columns.

The columns will have some backpres sure due to the bed of medium andthe fit(s). The backpres sure of a column will depend on the averagebead size, bead size distribution, average bed length, average crosssectional area of the bed, backpressure due to the frit and theviscosity and flow rate of the liquid passing through the bed.

Media

Because the invention is directed to the purification and/orconcentration of nucleic acids, extraction surfaces capable of adsorbingsuch molecules are particularly relevant. The chemistry employed in thepresent invention is typically based on normal phase or ion-exchange.Ion-pairing may also be used for nucleic acid purification. In general,these chemistries, methods of their use, appropriate solvents, etc. arewell known in the art.

The media or solid phase used in the column can be a form ofwater-insoluble particle (e.g., a porous or non-porous bead, fiber orother particle) that has an affinity for the nucleic acid of interest.Silica beads are suitable for the columns of the invention. Siliconquartz is large well. Davisil 923 and 635 work well. Chromosorb P alsoworks well. Other suitable materials include celite, diatomaceous earth,silica gel, silica gel, (Davisil, Impaq, Biotage), metal oxides andmixed metal oxides, glass, alumina, zeolites, titanium dioxide,zirconium dioxide. Ion exchangers made of inorganic or polymericsubstrates also work quite well.

The beads or particles used in the column have a shape or pore structurethat provides a large surface area or exposed surface. In someembodiments, the capture material has a surface area of greater than 0.5m², 1 m², 1.5 m², 2 m², 3 m², 5 m², 6 m², 10 m², 20 m² or 30 squaremeters per gram of material.

The bed volume of the medium used in the columns of the inventiondepends on the scale of the plasmid purification but it is typically inthe range shown in tables 2 and 4. In terms of percentage, the bedvolume can be in the range of 5% to 50% of the column volume. In certainembodiments, the bed volume can be in the range of 7%-40% of the columnvolume or in the range of 10%-30% of the column volume.

The space between resin particles can also be important. This spaceincreases with looser packing of the column. In certain embodiments, thecolumn beds are not tightly packed.

Frits

One or more frits is used to contain the bed of medium in a column.Frits can take a variety of forms, and can be constructed from a varietyof materials. The frits of the invention are porous, since it isnecessary for fluid to be able to pass through the frit. The frit shouldhave sufficient structural strength and integrity to contain theextraction media in the column. It is desirable that the frit havelittle or no affinity for chemicals with which it will come into contactduring the extraction process, particularly the analyte of interest.Frits of various pores sizes and pore densities may be used provided thefree flow of liquid and particulates is possible. Frits of pore sizelarge enough to prevent plugging from cell debris are of particularinterest. Some frits of the invention have a large pore size frit.

In one embodiment, a single frit (e.g., a lower, or bottom, frit)extends across the open channel of the column body. Often, the bottomfrit is attached at or near the open lower end of the column, e.g.,extending across the open lower end. This configuration is not required,i.e., in some embodiments, the bottom frit is located at some distanceup the column body from the open lower end. Normally, a bed of medium ispositioned inside the open channel in contact with the bottom frit.

In certain embodiments, a top frit may be employed. For example, in someembodiments, a second frit extends across the open channel between thebottom frit and the open upper end of the column body. In thisembodiment, the top frit, bottom frit and column body (i.e., the innersurface of the channel) define a media chamber wherein a bed of mediumis positioned. The fits should be securely attached to the column bodyand extend across the column body to completely occlude the channel,thereby substantially confining the bed of medium inside the mediachamber.

In some embodiments, the top frit can be just above the bed of medium orin contact with the bed of medium. In other embodiments, the top frit ispositioned well above the medium, e.g., 25 mm or more above the mediumin a 200 μl pipette tip column or 50 or more mm above the bed in a1.2-mL pipette-tip column. The position of the top frit can be proximalto open upper end of the pipette tip column. That is, the top frit canbe closer to the open upper end of the column than to the bed medium. Inthese embodiments, the bed is not packed and the medium can occupy wellunder 50% of the volume of the extraction media chamber and the top fritcan be significantly thicker than the bottom frit. In some embodiments,liquids may not flow through the top frit.

The position of the top frit over the bed may just touch the top of theresin bed or be positioned substantially above the resin bed. When thefrit is above the resin bed, the resin bed may move or expand withaspiration of liquids including the sample containing the particulates.The bed may move down against the bottom frit with expulsion of theliquid.

The performance of the column is typically enhanced by the use of fritshaving pore or mesh openings sufficiently large to allow cell debris orother particulates to flow through the frit without clogging or pluggingunder low pressures applied by a pipette or liquid handler. Of course,the pore or mesh openings of course should not be so large that they areunable to adequately contain the extraction media in the chamber. Fritsused on columns of the invention can have pore openings or mesh openingsof a size in the range of about 5-500 μm, more preferably 10-200 μm, andstill more preferably 100-150 μm, e.g., about 120 μm.

In some cases, it is necessary to consider the relationship between thefrit pore size and the particle diameter. Specifically, it is possibleto increase the frit pore size when the particle diameter is increased.For example, a frit pore size of 100 μm was used successfully with arange of different resins.

Some embodiments of the columns of the invention employ a thin frit,preferably less than 2000 μm in thickness (e.g., in the range of 20-2000μm, 40-350 μm, or 50-350 μm), more preferably less than 200 μm inthickness (e.g., in the range of 20-200 μm, 40-200 μm, or 50-200 μm),more preferably less than 100 μm in thickness (e.g., in the range of20-100 μm, 40-100 μm, or 50-100 μm). However, thicker frits, up toseveral mm, 5 and even 10 mm, thick may be used if the pore size of thefrit can be increased dramatically.

Some embodiments of the invention employ a membrane screen as the frit.The use of membrane screens as described herein typically provide thislow resistance to flow and hence better flow rates, reduced backpressureand minimal distortion of the medium. The membrane can be a woven ornon-woven mesh of fibers that may be a mesh weave, a random orientatedmat of fibers i.e. a “polymer paper,” a spun bonded mesh, an etched or“pore drilled” paper or membrane such as nuclear track etched membraneor an electrolytic mesh (see, e.g., U.S. Pat. No. 5,556,598). Themembrane may be, e.g., polymer, glass, or metal provided the membrane islow dead volume, allows movement of the sample and various processingliquids through the column bed, may be attached to the column body, isstrong enough to withstand the bed packing process, is strong enough tohold the column bed of beads, and does not interfere with the extractionprocess i.e. does not adsorb or denature the sample molecules.

The frit can be attached to the column body by any means which resultsin a stable attachment. For example, the screen can be bonded to thecolumn body through welding or gluing. The column body can be welded tothe frit by melting the body into the frit, or melting the frit into thebody, or both. Alternatively, a frit can be attached by a friction fitor by means of an annular pip, as described in U.S. Pat. No. 5,833,927.

The frits of the invention can be made from any material that has therequired physical properties described herein. Examples of suitablematerials include polymer, sintered polymer, fiber, nylon, polyester,polyamide, polycarbonate, cellulose, polyethylene, nitrocellulose,cellulose acetate, polyvinylidine difluoride, polytetrafluoroethylene(PTFE), polypropylene, polysulfone, PEEK, PVC, vinyl polymer, metal(e.g., steel), ceramic and glass.

In certain embodiments of the invention, a wad of fibrous material isincluded in the column, which extends across the open channel below theopen upper end of the column body, wherein the wad of fibrous materialand open channel define a media chamber, wherein the medium ispositioned within the media chamber. This wad of fiber can be a porousmaterial of glass, polymer, metal, or other material having large pores.In some embodiments, the wad of fibrous material is used in lieu of anupper frit.

Solvents

Disruption of bacterial cells is typically accomplished using analkaline solution containing a detergent. Any detergent that effectivelydisrupts the cell membrane can be used for this purpose. In otherembodiments, the lysis procedure is mechanical or physical. In somemethods, the lysis procedure involves treatment with a surfactant.

The lysis procedure is usually followed by the addition of aneutralizing solution (also known as a precipitation solution). Theneutralization solution may contain an acid. It may also contain achaotropic agent and/or other components.

In certain embodiments of the invention, chaotropic agents can be addedto the sample prior to nucleic acid capture. Examples of chaotropicreagents include sodium iodide, sodium perchlorate, guanidinethiocyanate (GuSCN), urea, guanidine hydrochloride (GuHCl), potassiumiodide, sodium perchlorate, potassium chloride, lithium chloride, sodiumchloride, urea or mixtures of such substances.

Examples of suitable solvents for use with the invention are shown inTables 5 and 6.

TABLE 5 Normal Phase Normal Phase Chaotropic Reverse Phase ExtractionExtraction Ion-Pair Extraction Typical solvent Low to medium High tomedium High to medium polarity range Typical sample Hexane, toluene,chaotropic buffers H₂O, buffers, ion- loading solvent CH₂Cl₂ alcoholpairing reagent Typical desorption Ethyl acetate, H₂O/buffer H₂O/CH₃OH,ion- solvent acetone, CH₃CN pairing reagent (Acetone, H₂O/CH₃CN, ion-acetonitrile, pairing reagent isopropanol, (Methanol, methanol, water,chloroform, acidic buffers) methanol, basic methanol, tetrahydrofuran,acetonitrile, acetone, ethyl acetate) Sample elution Least polar sampleMost polar sample Most polar sample selectivity components firstcomponents first components first Solvent change Increase solventDecrease Decrease solvent required to desorb polarity chaotropic bufferpolarity

TABLE 6 Hydrophobic Desorption Ion Exchange Interaction Affinity PhaseSolvent Features Extraction Extraction Extraction Typical solvent HighHigh High polarity range Typical sample H₂O, buffers H₂O, high salt H₂O,buffers loading solvent Typical desorption Buffers, salt H₂O, low saltH₂O, buffers, pH, solvent solutions competing reagents, heat, solventpolarity Sample elution Sample components Sample Non-binding, low-selectivity most weakly ionized components most binding, high-bindingfirst polar first Solvent change Increase ionic Decrease ionic ChangepH, add required to desorb strength or increase strength competingreagent, retained compounds change solvent pH or decrease pH polarity,increase heat

Methods for Using the Columns

The method involves capturing nucleic acids on pipette tip columns. Themethod can be performed in parallel and can be automated. Prior to thecapture step, the columns are usually wetted with an equilibrationsolution. After capture, the columns are washed to removenon-specifically bound material. Then the nucleic acids are releasedfrom the column in an elution step.

In certain embodiments of the method, the open upper end of the columnis operatively engaged with a pump and the sample, wash and/ordesorption solvents are aspirated and discharged through the open lowerend of the column. Often, aspirate/expel steps are repeated multipletimes, i.e., a plurality of in/out cycles can be employed to pass thesolvent back and forth through the bed more than once. In otherembodiments, fluids enter the column through the open upper end and exitthe column through the open lower end.

A pump can be used to pass liquids through the column. Alternatively,liquids can be passed through the column by gravity flow or vacuum. Inother embodiments, the capillary action can be used to wick solventsupward through the open lower end of the column.

The invention provides a pipette or pipettor (such as a multi-channelpipettor) suitable for acting as the pump in methods such as thosedescribed herein. In some embodiments, the pipettor comprises anelectrically driven actuator. The electrically driven actuator can becontrolled by a microprocessor, e.g., a programmable microprocessor. Invarious embodiments, the microprocessor can be either internal orexternal to the pipettor body.

In preferred embodiments of the invention, a plurality of columns isoperated in a parallel fashion, e.g., multiplexed. Multiplexing can beaccomplished, for example, by arranging the columns in parallel so thatfluid can be passed through them concurrently. When a pump is used tomanipulate fluids through the column, each column in the multiplex arraycan have its own pump, e.g., syringe pumps activated by a commonactuator. Alternatively, columns can be connected to a common pump, acommon vacuum device, or the like.

In certain embodiments the pipettor is a multi-channel pipettor. Inother embodiments, a robotic system such as those commercially availablefrom Dynamic Devices, Zymark, Hamilton, Beckman, Tecan, Packard, Matrix,PhyNexus, Agilent and others are used for nucleic acid purification. Insome embodiments, robots having a 96-channel pipetting head areutilized. In some embodiments, a robot with an 8-channel or a 12-channelpipetting head is used.

In certain embodiments, throughput is maximized by performing some stepswith bidirectional flow and other steps by unidirectional flow includingthe use of vacuum, pressure or gravity. For example, the capture stepcan be performed using bidirectional flow and the wash and elution stepscan be performed using vacuum or gravity flow. In these embodiments, thepipetting head of the robotic liquid handler can be utilized moreefficiently for simply dispensing liquids, allowing a greater number ofcolumns to be processed in parallel. It is also possible to perform thecapture, wash and elution steps using gravity or vacuum.

The invention also provides software for implementing the methods of theinvention. For example, the software can be programmed to controlmanipulation of solutions and addressing of columns into sample vials,collection vials, for spotting or introduction into some device forfurther processing.

During aspiration and expulsion, the lower end of the pipette tip columncan be positioned relatively close to the tube or well bottom, e.g.,within a range having a lower limit of about 0.05 mm, 0.1 mm, 0.2 mm,0.3 mm, 0.4 m, 0.5 mm, 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm from the bottomof the well, and an upper limit of 0.3 mm, 0.4 m, 0.5 mm, 1 mm, 2 mm, 3mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm or 10 mm of the well bottom. Forexample, in some embodiments the open lower end of a pipette tip columnis positioned with between 0.05 and 2 mm from a well bottom, or between0.1 and 1 mm from a well bottom. The term “well bottom” does notnecessarily refer to the absolute bottom of a well, but to the pointwhere the tip makes contact with the well when the tip is lowered to itsfull extent into the well, i.e., a point where the tip can seal with thewell surface. For example, in some microwell plate formats the wellstaper down to an inverted conical shape, so a typical tip column willnot be able to make contact with the absolute bottom of the well.

The invention also includes kits comprising one or more reagents and/orarticles for use in a process relating to solid-phase extraction, e.g.,buffers, standards, solutions, columns, sample containers, etc.

Nucleic acids and particularly plasmids can be purified from any sourceincluding eukaryotic or prokaryotic cells, tissues, body fluids (blood,serum, plasma, saliva, urine, feces), tissue culture, bacteria, viruses.The purification procedure can be used with low, medium or high copynumber plasmids. The instant invention can also be used to isolatenucleic acids from a gel.

The Drying Station

The methods of the instant invention utilize a drying station. Theterms, drying station, drying block, vacuum manifold and vacuum blockare used interchangeably herein. The drying station allows air to bepassed through the pipette tip columns. Some commercially-available DNApreparation methods utilize vacuum manifolds. However, these manifoldsare designed only to pull liquid through columns or plates. Commonformats for these manifolds are 24-column and 96-column capacity. Sincethese existing manifolds are open underneath the columns or plates, itis often necessary to monitor liquid flow through the different columnsto ensure the liquid has passed through all the columns. The dryingstation described herein differs from commercially available vacuummanifolds. While manifolds are designed mainly to pull liquid throughcolumns, the drying station allows prolonged airflow distributed evenlyover all the columns.

One embodiment of the drying station block is shown in FIGS. 2 and 3. Incontrast to existing manifolds, the channels within the block completelysurround each column. In certain embodiments, the channels surround eachcolumn over substantially the entire length of the column. This designensures liquid and air flow is comparable between columns.

In other embodiments, the drying station can be designed with a channelthat surrounds each column but not over the entire length to form a sealwith the pipette tip column, primarily at the lower end of the column.

Vacuum or airflow applied to the drying station can be controlled bysoftware so that it occurs at the appropriate time and for theappropriate duration.

FIGS. 2A-2C depict embodiments of the side and front views of the dryingstation and FIGS. 3A-3B show the top and bottom of the block. In thisembodiment, the block contains positions for 96 columns. In otherembodiments, the block may contain positions for any number of columnsincluding for example, 1, 2, 3, 4, 5, 6, 8, 12, 24, 48 or 384 columns.

FIG. 2A depicts an embodiment of the side view of the adapter block, thetops of eight columns 10 are inserted into top block 20. Top block 20 isseparated from bottom block 40 by sealing gasket 30. The gasket forms aseal around each individual column when they are inserted into the blockso that the airflow is applied through the columns and not around thesides of the column bodies. The bottom of bottom block 40 containsplastic lip 50. In this embodiment, the lip conforms to SBS standardizedformat for 96-well plates so that the base of the block can be insertedinto the vacuum manifold or any deck position of a robotic liquidhandler.

FIG. 2B depicts the front view of the drying station block pictured inFIG. 2A. It is identical to the side view shown in view A except thatthe row of twelve columns 10 can be seen.

FIG. 2C is a cut-away view of the drying station front view. Pipette tipcolumns 60 are exposed to show that when inserted into the block theyextend almost to the bottom. In this embodiment, the end of the columndoes reach the bottom of the vacuum block. In other embodiments, thelower ends of the columns will be even with the bottom of the dryingstation block. In still other embodiments, the ends of the columns willextend out past the base of the vacuum block. Opening 100 allows thevacuum to be applied at the bottom of the block and allows liquid andair passage through the columns sealed by gasket 30. Cross section oftop block 70 is separated from cross section of bottom block 80 bygasket 30. In certain embodiments, the column shape is frustoconical andthe holes at the interface of top block 70 and bottom block 80 have asmaller diameter than those on the upper surface of top block 70. Thenucleic acids are captured from the sample by column packing material90, and then washed and eluted.

FIG. 3 depicts and embodiment of the top view of the drying station andFIG. 3B shows the bottom view of the vacuum block. Lip 50 lies at thebottom of the block near bottom surface 130. Pipette tip columns areinserted into through holes 110 from top surface 120.

In an alternative design, the drying station block lacks a gasket. Evenwithout a gasket, the pipette tip columns are in sealing engagement withthe block. In this design, the block can be one solid piece or can becomprised of multiple pieces.

Genomic DNA Isolation

The method for purification of plasmid DNA from a cell culture wasmodified for purification of genomic DNA. Genomic DNA can be extractedand purified from any cell type including bacteria, plants and animals.Examples of different cell types include mouse tail, blood, saliva,tissues from biopsies, cerebrospinal fluid, animal tissues, plant tissueand whole organisms such as fruit flies, worm and embryos. Genomic DNAisolation can be performed in a completely automated fashion in 96-wellformat. The same automated procedure can also be used when fewer than 96samples are processed. Small footprint liquid handling systems such asthe PhyNexus MEA can perform the purification process.

To purify genomic DNA, the sample is transferred to individual wells ofa 96-well deep well plate in preparation for lysis. Typically, between 5and 100 mg of sample is used. Often, the amount of sample is between 10and 50 mg.

In certain embodiments, proteinase K can be added to the sample togenerate a more homogeneous sample. This is particularly advantageousfor tissue samples. Even with the addition of proteinase K, the sampleis still considered an unclarified lysate. After the addition ofproteinase K, the sample may contain particulates or it may be liquid.It is remarkable that genomic DNA can be isolated directly from a samplewithout removing the solid components.

After lysis, the sample can be incubated. In some embodiments, thesample is heated during the incubation step, while in other embodiments,incubation is performed at room temperature. Room temperature is definedhere in as between 20 and 30° C. In those embodiments in which thesample is heated, the incubation can be carried out at temperaturesbetween 35 and 100° C. More specifically, the incubation can beperformed at between 40 and 90° C. and often, the incubation isperformed at temperatures between 45 and 55° C.

When the incubation step is performed, the duration can be between 1 and20 hours. Liquid samples such as blood may require a shorter incubationperiod, in the range of 5-90 minutes. To perform a heated incubationusing automation, deep well plates can be moved from room temperaturepositions to heating positions using a robotic arm.

Next, RNase A can be added to each sample and the samples can beincubated. Again, the samples can be incubated at room temperature or, ahigher or lower temperature can be used. Incubation times can be veryshort, (minutes) or longer (hours).

Bacterial samples can be lysed as described above. For tissue samples,lysis can be carried out using similar reagents and conditions to thoseused for bacterial cells. Among other ingredients, lysis buffers cancontain buffers such as Tris, salts (e.g., NaCl), chelators such asEDTA, and detergents such as SDS and surfactants such as CTAB(Cetyltrimethylammonium bromide). In some embodiments, two differentlysis buffers can be used in sequence. After addition of the lysisbuffer, the samples can be incubated for varying times and temperatures,depending on the sample type. For example, the samples can be incubatedat 50-90° C. for 5-30 minutes. In other embodiments, the sample is notheated and is incubated at room temperature. Prior to loading the sampleon the column, an alcohol such as ethanol can be added to the samples.

There is no requirement for centrifugation and the viscous samples canbe quite viscous. Nevertheless, they can be purified with pipette tipcolumns. Unlike the procedure for purification of plasmid DNA, thegenomic preparation procedure does not require a precipitation step. Thegenomic DNA must be kept in solution in order to extract it with thepipette tip columns. In the plasmid purification method described above,the genomic DNA is precipitated to remove it from solution. However, inthe genomic DNA method, the precipitation buffers are omitted to keepthe genomic DNA accessible for the pipette tip column.

The pipette tip columns can be equilibrated on the head of the MEA oranother instrument. The pipette tip columns can then be equilibratedwith water or buffer.

To capture genomic DNA, the pipette tip columns can be unloaded into adrying station on the MEA instrument. The MEA can then use pipette tipsto add the sample to the top of the resin bed and passage of the samplethrough the pipette tip column can be achieved by gravity or by applyinga vacuum. In alternative embodiments, the sample is aspirated throughthe open lower end of the pipette tip column and the capture step can beperformed with back and forth flow.

Depending on the sample type, it may be desirable to perform incubationsteps at different stages during the purification procedure as describedabove. For example, an incubation can be performed after treatment withproteinase K, after lysis, or after treatment with RNase A. Incubationscan be performed at an appropriate temperature and for an appropriateduration.

After the capture step, a wash step can be performed although in someembodiments, the wash step is omitted. Wash buffers are typicallycomprised of water, buffers, alcohols and combinations thereof. As withthe capture step, the wash can be performed with unidirectional orbidirectional flow. The pipette tip columns can be loaded onto the MEApipetting head and washed using bidirectional flow. In some methods apause is incorporated after each aspirate step and/or after eachexpulsion. The alcohol concentration can be in the range of 60-100%. Thewash can be repeated with fresh wash buffer for one, two or moreadditional washes.

After the wash, the columns can be transferred to the drying station andalcohol is removed from the bed of the pipette tip columns by flowing1-500 cubic liters of air through the pipette tip column. The genomicDNA can then be eluted in a volume of elution buffer, typically in therange of 50-500 μL. The elution buffer can be aspirated into the columnthrough the lower end or added from the upper end of the column. Theelution buffer is typically comprised of water or buffer. In certainembodiments, the elution buffer is incubated on the column for severalminutes prior to dispensing the purified DNA. For example, theincubation step can be performed for a period of between 1 and 20minutes.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration, and are not intended to be limitingof the present invention, unless so specified.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and practice the presentinvention. They should not be construed as limiting the scope of theinvention, but merely as being illustrative and representative thereof.

Example 1

Evaluation of an 80 μL Bed Volume Pipette Tip Column Containing a Resinfor Purification of Plasmid from Eukaryotic Cells

In this example, the performance of 80 μL bed volume pipette tip columnsis evaluated. The pipette tip column was constructed from a 200 μLpipette tip (Tecan) and is packed with a silica-based particle resin.These columns, buffer conditions and column processing procedures aretested for the recovery of plasmid DNA from yeast. The yield and qualityare assessed by UV spectrometry and agarose gel electrophoresis.

Samples are prepared by growing a single yeast colony in 25 mL mediumsupplemented with the appropriate carbon source to propagate the DNAvector. The liquid culture is incubated at 30° C. with shaking until theculture becomes turbid. The culture is divided into equal aliquots andsubjected to centrifugation at 5,000×g for 15 minutes to pellet theyeast. The supernatant is discarded and the pellets are lysed by mortarand pestle, using liquid nitrogen and resuspended in buffer.

To purify the plasmid DNA from the lysed yeast cells, the pipette tipcolumns are processed by the ME semi-automated purification system(PhyNexus, Inc., San Jose, Calif.). The columns are equilibrated with200 μL 7M guanidinium-HCl by performing one cycle of back-and-forth flowat 500 μL/min and a 20 second pause at the end of the aspirate anddispense steps.

The yeast lysate is subjected to pipette tip column processing forcapture of the plasmid DNA by using at least 24 back-and-forth cycles ata flow rate of 250 μL/min with 20 second pauses after the end of eachaspirate and dispense step.

Following plasmid capture on the pipette tip column, the columns arewashed with 200 μL wash 1 buffer consisting of 10 mM Tris-HCl pH 6.6, 5Mguanidinium-HCl and 30% ethanol. This is followed by a second wash inwash 2 buffer consisting of 10 mM Tris-HCl pH 7.5 and 80% ethanol. Bothwash procedures are carried out using one cycle of back-and-forth flowat a flow rate of 500 μL/min with 20 second pauses at the end of eachaspirate and expel step. A blow out step is incorporated to remove allresidual wash buffer from the resin bed.

DNA plasmid is released from the column with 300 μL elution bufferconsisting of water. The procedure to release the DNA is 8back-and-forth cycles at a flow rate of 250 μL/min with 20 second pausesafter the end of each aspirate and dispense step.

Example 2

Purification of Plasmid DNA from E. coli

Columns and methods for purifying plasmid DNA from E. coli lysate weredeveloped for 96 samples at a time. The columns used in this examplewere 80 μL bed columns fitted with 100 μm pore size screen bottom fits.The method was designed to operate on a Tecan EVO, Biomek FX or otherrobotic liquid handler. The solutions used are listed in Table 7.

E. coli cells were grown to late logarithmic phase, harvested bycentrifugation and then resuspended in buffer. The plasmid purificationprocedure developed was as follows.

-   1. Add 250 μL of Lysis buffer to resuspended cells using gentle    pipette mixing for 3 minutes.-   2. Add 350 μL of Neutralization buffer to lysed culture using gentle    pipette mixing for 3 minutes.-   3. Attach plasmid DNA pipette tip columns to 96 channel head.-   4. Equilibrate the pipette tip columns by cycling through the    equilibration buffer.    -   Use 2 cycles at 0.5 mL/min flow rate.-   5. Capture the plasmid DNA.    -   Use 24 cycles at 0.25 mL/min flow rate.-   6. Wash (Washl buffer, 500 μL) the captured plasmid DNA.    -   Use 2 cycles at 0.5 μL/min flow rate.-   7. Wash (Wash2 buffer, 500 μL) the captured plasmid DNA.    -   Use 2 cycles at 0.5 μL/min flow rate.-   8. Wash (Wash2 buffer, 500 μL) the captured plasmid DNA.    -   Use 2 cycles at 0.5 mL/min flow rate.-   9. Blowout remaining wash buffer.-   10. Elute the captured plasmid DNA.    -   Use 16 cycles at 0.25 mL/min flow rate.

The yield was approximately 5 μg per well. The purity was examined withagarose gel electrophoresis and UV absorption with A₂₆₀/A₂₈₀ ratiobetween 1.8 and 2.0.

Example 3

Purification of Plasmid DNA from E. coli Pellets

Columns and methods for purifying plasmid DNA from E. coli lysate weredeveloped for 96 samples at a time. The columns used in this examplewere 80 μL bed columns fitted with 100 μm pore size screen bottom fits.The method was designed to operate on a Tecan EVO, Biomek FX or otherrobotic liquid handler. The solutions used are listed in Table 7.

E. coli cells were grown to late logarithmic phase, harvested bycentrifugation and then resuspended in 150 μL Resuspension Buffer (50 mMTris-HCl pH 8.0, 10 mM EDTA, 400 μg/mL RNase A). The plasmidpurification procedure was performed as follows.

Using wide bore pipette tips, 150 μL of Lysis buffer (200 mM NaOH, 1%SDS) was added to the resuspended cells using gentle pipette mixing.Next, the precipitation step was carried out by the addition of 210 μLof Precipitation Buffer (0.9 M potassium acetate pH 4.8, 4.2 Mguanidinium hydrochloride) to lysed cells using gentle pipette mixing.The wide bore pipette tips were discarded and the liquid handling robotattached plasmid DNA pipette tip columns to the 96-channel head. Thepipette tip columns were equilibrated in 500 μL of water withback-and-forth cycling of the equilibration buffer. A typical cycleconsists of aspiration of 180 μL at a flow rate of 0.5 mL/minutefollowed by a pause of about 30 seconds while maintaining the end of theplasmid DNA pipette tip columns at the bottom of the well of a deep wellor microplate. The second half of a cycle consists of dispense of 180 μLat a flow rate of 0.5 mL/minute followed by a pause of about 30 secondswhile maintaining the end of the plasmid DNA pipette tip columns at thebottom of the well of a deep well or microplate. The plasmid DNA pipettetip columns next capture plasmid from the prepared E. coli samples using14cycles at 0.25 mL/min flow rate with 20 second pauses. After plasmidcapture, the plasmid DNA pipette tip columns were blotted onto absorbentlab paper by to remove cell debris and precipitants. The DNA pipette tipcolumns next went through wash by submerging the end of the column on500 μL of Wash Buffer (100 mM Tris-HCl pH 7.5, 65% ethanol) using 2cycles at 0.5 μL/min flow rate and 20 second pauses. Wash was repeatedtwice in 500 μL of fresh Wash Buffer. The ethanol was dried from theresin bed by ejecting the plasmid DNA pipette tip columns in a vacuumadapter and a vacuum was applied that was capable of moving at least 4Cubic Feet per Minute (CFM). The vacuum was applied for 5 minutes. Theliquid handling robot reattached the plasmid DNA pipette tip columns andeluted the pure plasmid DNA by aspirating 170 μL Elution Buffer (10 mMTris-HCl pH 8.5) and incubating it for 5 minutes. The plasmid wasreleased by dispensing 170 μL into a microplate.

Example 4 Comparison of Pipette Tip Columns and Spin Columns

The pipette tip columns used in this example contained 80 μL ofChromosorb P resin (Sigma Aldrich) and were fitted with 105 μm pore sizescreen bottom frits. A side by side comparison with commercial spincolumns was made using buffers listed in Table 7. E. coli was grownovernight in 1.4 mL medium in a 96-well deep-well plate. The results ofthree representative samples are shown in Table 5.

TABLE 7 Buffers Buffer Name Content Resuspension 50 mM Tris-HCl pH 8.0,10 mM buffer EDTA, 100 ug/mL RNase A Lysis buffer 200 mM NaOH, 1% SDSNeutralization 4.2M guanidine hydrochloride buffer 0.9M Potassiumacetate pH 4.5 Equilibration water buffer Wash1 buffer 5M guanidinehydrochloride 30% Ethanol, 10 mM TRIS-HCl pH 6.6 Wash2 buffer 10 mMTRIS-HCl pH 7.5, 80% Ethanol Elution buffer Water

TABLE 8 Comparison of pipette tip columns and spin columnsRepresentative results from purification of plasmid performed with acommercial spin columns used with three sequential elutions (Spin C1,Spin C2 and Spin C3) and two types of pipette tip columns. E1, E2 and E3refer to the recovery from three sequential elution aliquots, elutions 1through 3. Conc Combined Column Name A260 (ng/μL) A260/A280 Total μgtotal μg Spin C1 (100 μL 0.86 43.05 1.78 4.30 elution) Spin C2 (100 μL0.19 9.6 1.63 0.96 elution) Spin C3 (100 μL 0.14 7.05 1.10 0.70 5.97elution) Pipette Tip [top frit] 1.12 56.2 1.97 5.62 E1 (100 μL) PipetteTip [top frit] 0.51 25.5 1.93 2.55 E2 (100 μL) Pipette Tip [top frit]0.28 14.25 1.73 1.42 9.59 E3 (100 μL) Pipette Tip [no top 0.61 30.751.92 3.07 frit] E1 (100 μL) Pipette Tip [no top 0.64 31.95 1.92 3.19frit] E2 (100 μL) Pipette Tip no [top 0.37 18.55 1.80 1.85 8.12 frit] E3(100 μL)

Example 5

Miniprep of E. coli Plasmid DNA from 96 Samples at a Time

Single colonies were inoculated into 1.4 ml rich medium (containing theappropriate antibiotic) in a 2-ml deep-well block and incubated at 37°C. and 300 rpm for 16 hours. The deep-well block was centrifuged and themedium was discarded. The plate was then transferred to a Tecan FreedomEvo liquid handler with the deck set up described as follows and shownin FIG. 4.

Positions 1 through 3 contain boxes of 200-μL pipette tips. Position 4has a box of 96 pipette tip columns. In this example the pipette tipcolumns are constructed with a bottom frit only (pore size 105 μm) andfilled with 80 μL of silica resin. Position 5 holds a 96-well platefilled with 250 μL Precipitation Buffer in each well. Positions 6 and 7contain plates holding lysis and resuspension buffers, respectively.Positions 8, 11 and 12 contain buffers for wash 1, wash 2 and wash 3,respectively. In this procedure, wash 3 is the same solution as wash 2.Each of these is a deep-well block holding 500 μL of buffer. A deep-wellblock holding 300 μL Equilibration buffer is placed in position 9. Thedeep-well plate holding the cell pellets is placed at position 10. Thereis a UV-readable plate at position 13 to receive the purified plasmidDNA. Stations 14 and 15 can be used for drawing air through the pipettetip columns with vacuum and a UV plate reader resides at position 16.

-   The plate was processed as follows.-   1. Resuspend cells. Transfer 150 ul resuspension buffer to cell    pellet. 130 ul, 8-16 cycles, 10 ml/min.-   2. Lyse cells. Add 150 uL of Lysis buffer to resuspended cells. 8    cycles of 180 μL at 10 ml/min with 2 sec pause.-   3. Add 210 μL precipitation buffer. 8 cycles of 180 μL at 10 ml/min    with 2 sec pause.-   4. Attach pipette tip columns to the 96-channel head. Equilibrate    the pipette tip columns. 2 cycles of 180 μL, 0.5 ml/min with 5 sec    pause.-   5. Capture    -   a. Aspirate 200 μL air at 0.25 ml/min with 2 sec pause    -   b. Submerge pipette tip column in unclarified lysate and expel        200 μL air at 0.25 ml/min with 2 sec pause. Particulates should        float.    -   c. Capture. 180 μL of unclarified lysate, 14 cycles at 0.25        ml/min with 20 sec pause.-   6. Wash 1. 180 μL of wash buffer 1,2 cycles at 0.5 ml/min with 10    sec pause.-   7. Wash 2. 180 μL of wash buffer 2, 2 cycles at 0.5 ml/min with 10    sec pause.-   8. Wash 3. 180 μL of wash buffer 2, 2 cycles at 0.5 ml/min with 10    sec pause.-   9. Vacuum dry. Deposit tips to vacuum station and vacuum air through    the tips for 5 min.-   10. Elution    -   a. Aspirate 70 μL of air    -   b. Engage tips and aspirate 130 μL of elution buffer at 0.25        ml/min.    -   c. Incubate 5 min.    -   d. Expel 130 μL of purified plasmid at 0.25 ml/min.

TABLE 9 Solutions Buffer Name Content Resuspension 50 mM Tris-HCl pH8.0, 10 mM buffer EDTA, 0.4 mg/mL RNase A Lysis buffer 200 mM NaOH, 2.5%SDS Precipitation (A) 0.9M Potassium acetate pH 4.5 buffer (B) 4.2MGuanidinium-HCl Equilibration Water Solution Wash buffer 100 mM TRIS-HClpH 7.5, 65% Ethanol Elution buffer 10 mM Tris, pH 8.5

Example 6

Procedure for Midiprep of E. coli Plasmid DNA from 96 Samples at a Time

The buffers used in this example are listed in Table 9.

-   1) In 10 mL of LB or Agencourt Ale medium, inoculate a single    colony.-   2) Grow overnight. 37° C., 16 hours at 300 rpm.-   3) Centrifuge for 25 minutes at 3000 rpm.-   4) Discard the supernatant.-   5) Resuspend pellet with 150 μl Resuspension buffer.-   6) Add 1 mL of Lysis buffer. Mix thoroughly.-   7) Add 1.4 mL of Precipitation buffer. Mix thoroughly.-   8) Attach pipette tip columns to the ME/MEA and equilibrate in 500    uL of Equilibration solution. The columns contain a 300 μL bed in a    1 mL pipette tip (2 cycles at 0.5 ml/min)-   9) Intake 1 mL air into the column at a flow rate of 0.5 ml/min.-   10) Move the pipette tip column to the bottom of the precipitated    sample.-   11) Expel 1 mL of air at 10 ml/min.-   12) Capture plasmid by performing 10-15 cycles (0.25 ml/min or 0.5    ml/min).-   13) Five wash steps. Move the pipette tip columns into a deep well    block containing 1 mL of wash buffer. 4 cycles (0.5-5 ml/min).-   14) Air dry. Use vacuum pump. 5-15 minutes.-   15) Move the pipette tip columns into the deep well block containing    elution buffer.-   16) Intake 1333 μl, wait 5 min and expel.

Example 7

Midiprep of E. coli Plasmid DNA using a Combination of Back and ForthFlow and Gravity Flow.

In this example, the midiprep is performed as described in the precedingexample except the wash and elution steps are done using gravity flow.The column is washed with 1 ml of buffer and the wash step is repeated10-15 times. For the elution step, 1.2 mL of elution buffer is used.

Example 8

Midiprep of E. coli Plasmid DNA using Gravity Flow.

In this example, the midiprep is performed as described in the precedingexample except the capture step is performed using gravity flow.

Example 9

Extraction of DNA from Agarose Gel

The nucleic acids in this example are not limited to plasmid DNA. Thisprocedure can be used to isolate nucleic acids of any type or sizedistribution that can be visualized on a gel. Agarose gelelectrophoresis is the most common method for size separation andvisualization of double stranded DNA. Agarose gels are used to separateDNA based on the mass (and thus, the length) of the DNA. Shorter DNAmigrates farther through the gel compared to a long DNA. In practice,agarose gels are used to purify PCR products away from free primers,dNTPs, DNA polymerase and buffer components. The PCR product willmigrate as a discreet band. Restriction digests of plasmids, forexample, also result in discreet bands that can be purified by agarosegel. Discreet bands correspond to DNA of the same length. To utilizethis separation as a pre-purification tool, the band corresponding tothe DNA length of interest is excised from the gel using a scalpel orrazor blade. The band is weighed and is placed into a microfuge tube.Three volumes of gel extraction buffer (50 mM MOPS pH 7.0, 1M NaCl, 15%(v/v) isopropanol) is added to the excised gel using the conversion 1mg=1 μL. The tube is incubated at 50° C. for 10 minutes. The tube isvortexed every 2 to 3 minutes during this incubation. One volume ofisopropanol is added to the tube.

A plasmid DNA pipette tip purification column is used to capture theDNA. The column is processed by the PhyNexus MEA personal purificationinstrument. The MEA engages the pipette tip column and equilibrates itwith 2 cycles of back-and-forth flow in water using a flow rate of 0.5mL/min and 20 second pauses at the end of each aspirate and dispensestep. Next, the column captures the extracted DNA. This is accomplishedusing 4-20 cycles of back-and-forth flow at a flow rate of 0.25 mL/minand 20 second pauses at the end of each aspirate and dispense step. Thecolumns are subject to a wash in 0.5 mL Wash Buffer (80% ethanol, 10 mMTris-HCl pH 7.5). The wash is repeated in fresh buffer an additional twotimes. After washing, the pipette tip columns are transferred to avacuum block and subject to 5 minutes of vacuuming to dry the columns toremove residual Wash Buffer components. The MEA next engages the pipettetip columns and aspirates 130 μL of water and incubates for 5 minutes.This is dispensed to release the plasmid DNA and a second elution isperformed if necessary.

Example 10 Automated Purification of Bacterial Genomic DNA on thePhyNexus MEA

-   1. Grow bacterial cells in 25 to 50 mL Agencourt Ale in 250 mL shake    flasks overnight at 37° C.-   2. Aliquot cells (less than 1 ml per well) into 96-well, deep-well    plates.-   3. Lyse with 1000 μL Lysis Buffer (9 mM Tris, 0.55% SDS—final pH 9)-   4. Mix by pipetting back/forth-   5. Incubate 30 min at room temperature-   6. Add 100 μL PL buffer (20 mM EDTA; 3.885 μg/μL RNaseA)-   7. Mix by pipetting back/forth-   8. Capture unidirectionally with assistance by vacuuming and    agitation by engaging columns and executing quick back and forth    pipetting. Capture: 500 μL of the lysate is taken up and dispensed    on the top of the column. The vacuum is engaged for 30 seconds. The    columns are engaged and quick (20 mL/min) back and forth pipetting    of 500 μL volume is carried out to loosen the resin bed. The columns    are disengaged back into the vacuum and vacuum for 30 seconds. This    whole process is repeated two more times with the rest of the    lysate.-   9. Repeat until all lysate is processed through bed-   10. Wash by cycling 5×1 mL WB(P)75 (100 mM Tris pH 7.5, 75% EtOH)    with vacuuming for 30 seconds between each aliquot-   11. Vacuum for 5 minutes to evaporate ethanol-   12. Pump 280 μL EB(P) (10 mM Tris pH 8.5) back and forth for 5    cycles with pauses after intakes totaling 8 minutes.    The PhyNexus MEA method is described in detail below.-   1. Resuspend cell pellet with 200 μL of resuspension buffer.-   2. Add 1 mL of lysis buffer to resuspended sample.-   3. Mix by aspirating and expelling 1 mL for 15 cycles at 13.3    mL/min.-   4. Incubate sample at room temperature for 30 minutes.-   5. Add 100 μL of PL buffer.-   6. Mix by intaking and expelling 1 mL for 2 cycles at 13.3 mL/min.-   7. The columns contain an 80 μL bed in a 1 mL pipette tip. Attach    the pipette tip columns to the PhyNexus MEA and equilibrate in 500    μL of Equilibration solution (3 cycles at 13.3 mL/min).-   8. Move columns into the drying station.-   9. Intake 500 μL of sample and dispense the sample into the pipette    tip columns.-   10. Vacuum the columns for 30 seconds.-   11. Engage the pipette tip columns onto pipette head and quickly    intake and expel 500 μL of air for 5 cycles at 20 mL/min to loosen    the resin bed.-   12. Disengage the columns into the drying station.-   13. Pass vacuum through the samples for 30 seconds.-   14. Repeat steps 9 through 13 two more times.-   15. Engage the pipette tip columns and move the columns to bottom of    a well containing 1 mL of wash buffer.-   16. Intake and expel 900 μL of the wash buffer at 1 mL/min for 2    cycles with 10 second pause after each intake and expel.-   17. Disengage the columns into the drying station.-   18. Pass air through the columns for 30 seconds.-   19. Engage the pipette tip columns and move them to the bottom of a    second well containing 1 mL of fresh wash buffer.-   20. Intake and expel 900 μL of the wash buffer at 1 mL/min for 2    cycles with 10 second pause after each intake and expel.-   21. Disengage the columns into the drying block.-   22. Pass vacuum through the columns for 30 seconds.-   23. Engage the pipette tip columns and move the columns to the    bottom of a third well containing 1 mL of fresh wash buffer.-   24. Intake and expel 900 μL of the wash buffer at 1 mL/min for 2    cycles with 10 second pause after each intake and expel.-   25. Repeat steps 23 and 24 with two more 1 mL aliquots of fresh wash    buffer.-   26. Disengage the columns into the drying station.-   27. Pass air through the columns for 5 minutes.-   28. Engage the pipette tip columns and move them to the bottom of a    well containing 280 μL of Elution buffer.-   29. Intake and expel 280 μL of the elution buffer into the well that    contained the elution buffer. The flow rate is 0.5 mL/min for 2    cycles with 60 second pause after each intake.-   30. Position the columns above the eluted sample surface and expel    the remaining liquid from the column.

Example 11 Automated Purification of Worm DNA on the PhyNexus MEA

-   The method for purification of plasmid DNA from culture was modified    for purification of genomic DNA from tissues. Generally, the    modifications include the following changes-   1. The preparation of sample. The tissues were prepared to generate    a viscous supernatant by dissolving the solid tissues.-   2. The precipitation procedure was eliminated because genomic DNA    must stay in solution.-   3. The capture of the sample was carried out by a single pass    through the pipette tip column.-   4. The stringency of the wash buffer was decreased to reduce loss of    genomic DNA.

The following procedure was used for purification of genomic DNA fromwhole blood worms, Chironomidae tetans.

Procedure:

-   1) Transfer 20 mg sample to individual wells of a deep well plate.-   2) Add 20 μL of 600 mU/mL Proteinase K to each sample.-   3) Add 180 μL Lysis Buffer 1A (50 mM Tris-HCl pH 8.0, 100 mM NaCl,    3% SDS) to each sample. Note: Lysis Buffer 1B (100 mM Tris-HCl pH    8.0, 1.4 M NaCl, 20 mM EDTA, 3% CTAB (Cetyltrimethylammonium    bromide)) can alternatively be used for lysis.-   4) Incubate the deep well plate at 56° C. for 16 hours.-   5) Add 5.7 μL of 0.07 mg/mL RNase A.-   6) Incubate at room temperature for two minutes.-   7) Add 200 μL of Lysis Buffer 2 (Qiagen ATL Buffer).-   8) Incubate at 70° C. for ten minutes.-   9) Add 200 μL of 100% ethanol to each sample.-   10) Equilibrate pipette tip columns with 500 μL deionized water    using two cycles of back-and-forth flow at a flow rate of 13 mL/min    and 10 second pauses at the end of each aspirate and each dispense.-   11) Capture genomic DNA by loading the sample to the top of the    resin bed and flow through the pipette tip column by gravity or    using vacuum.-   12) Wash with 500 μL of Wash Buffer (10 mM Tris HCl pH 7.4, 70%    Ethanol)-   13) consisting of 2 cycles at a flow rate of 0.5 mL/minutes and 20    second pauses at the end of each aspirate and each dispense-   14) Repeat step 12 twice using fresh Wash Buffer.-   15) Dry the ethanol from the resin bed by flowing 2.5 liters of air    through the pipette tip column.-   16) Elute the genomic DNA by aspirating 210 μL Elution Buffer (10 mM    Tris HCl pH 8.5), incubating for 5 minutes and dispensing to release    the purified DNA.

Example 12 Automated Purification of Mouse Tail DNA on the PhyNexus MEA

Mouse tail DNA is purified exactly as described above for worm DNA.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover and variations,uses, or adaptations of the invention that follow, in general, theprinciples of the invention, including such departures from the presentdisclosure as come within known or customary practice within the art towhich the invention pertains and as may be applied to the essentialfeatures hereinbefore set forth. Moreover, the fact that certain aspectsof the invention are pointed out as preferred embodiments is notintended to in any way limit the invention to such preferredembodiments.

What is claimed is:
 1. A method for capturing plasmid DNA on a column,comprised of: a) providing a column, wherein the column is comprised ofa solid phase; b) providing a cell lysate comprised of a liquid and aflocculent, wherein the liquid contains plasmid DNA; c) providing alarge-pore filter having a pore size, wherein the pore size of thelarge-pore filter is greater than 50 μm; d) passing the cell lysatethrough the large-pore filter to produce a partially clarified lysate;and e) passing the partially clarified lysate over the column, wherein aportion of the plasmid DNA is captured on the solid phase.
 2. The methodof claim 1, wherein the pore size is greater than 200 μm
 3. The methodof claim 1, wherein the partially clarified lysate is comprised ofbetween 3% and 40% (vol/vol) of the flocculent
 4. The method of claim 1,wherein the solid phase is comprised of silica or an anion exchangeresin.
 5. The method of claim 1, wherein the cell lysate is comprised ofa chaotropic solution.
 6. The method of claim 1, wherein step (e) isautomated.
 7. The method of claim 8, wherein step (d) is automated. 8.The method of claim 1, wherein step (d) is performed using gravity flow.9. The method of claim 1, wherein step (d) is performed using vacuum orpressure.
 10. The method of claim 1, wherein the following steps areperformed after step (e): f) passing a wash buffer through the column,and g) eluting the plasmid DNA by passing a elution buffer through thecolumn, wherein the eluted plasmid DNA is a substantiallyendotoxin-free.
 11. The method of claim 13, wherein the eluted plasmidDNA has an endotoxin concentration of less than 50 endotoxin units permicrogram.
 12. A method for capturing plasmid DNA on a column, comprisedof: a) providing a column, wherein the column is comprised of a solidphase; b) providing a cell lysate comprised of a liquid and aflocculent, wherein the liquid contains plasmid DNA; c) providing alarge-pore filter having a pore size; d) passing the cell lysate throughthe large-pore filter to produce a partially clarified lysate, whereinthe partially clarified lysate is comprised of between 3% and 40%(vol/vol) of the flocculent; and e) passing the partially clarifiedlysate over the column, wherein a portion of the plasmid DNA is capturedon the solid phase.
 13. The method of claim 12, wherein the pore size isgreater than 200 μm
 14. The method of claim 12, wherein the partiallyclarified lysate is comprised of between 3% and 40% (vol/vol) of theflocculent
 15. The method of claim 12, wherein the solid phase iscomprised of silica or an anion exchange resin.
 16. The method of claim12, wherein step (e) is automated.
 17. The method of claim 12, whereinstep (d) is performed using gravity flow.
 18. The method of claim 12,wherein step (d) is performed using vacuum or pressure.
 19. The methodof claim 12, wherein the following steps are performed after step (e):a) passing a wash buffer through the column, and b) eluting the plasmidDNA by passing a elution buffer through the column, wherein the elutedplasmid DNA is a substantially endotoxin-free.
 20. The method of claim19, wherein the eluted plasmid DNA has an endotoxin concentration ofless than 50 endotoxin units per microgram.